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Antimicrobial Agents and Chemotherapy, March 2001, p. 727-733, Vol. 45, No. 3
Department of Medical Microbiology and
Immunology, University of Alberta, Edmonton, Alberta, Canada
Received 5 September 2000/Returned for modification 15 November
2000/Accepted 7 December 2000
In this study, we systematically examined in vitro frequencies and
spectra of the spontaneous mutations in Helicobacter
pylori that confer resistance to clarithromycin
(Clar), metronidazole (Mtzr), amoxicillin
(Amxr), ciprofloxacin (Cipr), and rifampin
(Rifr). The mutation rate of Rifr
or Cipr determined in a fluctuation assay is
1 × 10 Antibiotic resistance is an
increasing problem for the treatment of infectious diseases. Bacteria
have evolved diverse mechanisms (pathways) of resistance to
antimicrobial agents, including control of uptake and efflux of
drugs, modification and detoxification of drugs, alteration and
protection of the target sites, and acquisition of heterologous
resistance genes from external sources. In Helicobacter pylori, the etiological agent of a wide range of gastric diseases, genetic determinants for resistance to several antibiotics, including clarithromycin, metronidazole, ciprofloxacin, and rifampin, have been
determined. Remarkably, the known mechanisms of antibiotic resistance
in H. pylori are all due to mutations in chromosomal genes.
Clarithromycin resistance is associated with mutations in the 23S rRNA
gene (22, 25), which inhibit the binding of clarithromycin
to the ribosome. Ciprofloxacin resistance is due to mutations in the
gyrA gene, which encodes the A subunit of DNA gyrase
(16), and rifampin resistance results from mutations in
the rpoB gene, encoding the The importance of de novo mutation in developing antibiotic resistance
prompted us to ask how mutations occur in H. pylori (28). The first step in elucidating the mechanisms of
mutagenesis is to define the background of frequency and specificity of
spontaneous mutations. From the pioneering works of Luria and Delbruck
(14) and recent developments in determining mutation
rates of bacterial populations, it is known that determination of
mutation rates is not simple (15, 17), and determination
of mutation spectra is particularly tedious. In this study we
systematically examined the in vitro frequencies and spectra of
spontaneous mutations in H. pylori that confer
resistance to clarithromycin, metronidazole, amoxicillin,
ciprofloxacin, and rifampin.
H. pylori strains, growth medium, and
antibiotics.
H. pylori reference strains
26695, NCTC11639, and UA802 (26), as well as some isolates
from University of Alberta Hospital, were used; all are
susceptible to the antibiotics tested in this study. H.
pylori strains were grown on BHI-YE broth (3.7% brain heart
infusion with 0.3% yeast extract and 5% animal serum) or agar plates
at 37°C under microaerobic conditions (5% CO2, 5% H2, and 90% N2). Antibiotics used in
this study include clarithromycin (Bayer), metronidazole (Sigma),
ciprofloxacin (Bayer), rifampin (Sigma), and amoxicillin (Sigma).
MIC test.
H. pylori cells were grown for 2 days and suspended in sterile BHI-YE liquid medium, and the turbidity
of the suspensions was adjusted to that of a 2.0 McFarland standard.
The suspended cells were inoculated (8 µl/spot) onto BHI-YE agar
plates containing different concentrations of antibiotics obtained by
serial twofold dilution. The plates were incubated as described above,
and the growth was examined after 3 days.
Determination of mutant frequency and mutation rate.
An
H. pylori strain that is susceptible to an antibiotic
was grown in BHI-YE broth to late log phase (about 3 days), with the
viable cell number being around 109/ml. This
culture was diluted 10 Selection and identification of spontaneous mutations.
Independent cultures were grown and plated as described above. To
ensure that all mutations represent independent events (but are not the
descendents of the same mutation), only one mutant colony was picked
from each culture. From the selected independent mutants, a DNA
fragment containing the respective gene of interest was PCR amplified
using the primers listed in Table 1. By
DNA sequencing, the mutations in the gene that are responsible for resistance were identified by comparison with the nucleotide sequence of the wild-type susceptible strain. When a distinct mutation in the
gene was identified, it was designated a specific allele of that gene
(i.e., a specific variant of the gene that conferred resistance).
0066-4804/01/$04.00+0 DOI: 10.1128/AAC.45.3.727-733.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Spontaneous Mutations That Confer Antibiotic Resistance in
Helicobacter pylori
ABSTRACT
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Abstract
Introduction
Materials and Methods
Results and Discussion
References
8 to 2 × 108 per
cell per division. In contrast, the mutation rates of Clar,
Mtzr, and Amxr are much
lower (<109). However, Mtzr mutants
could be readily selected in vitro by using the serial passage
method, suggesting that the mutagenic effect and selective effect of a sublethal dose of metronidazole contribute to the rapid
development of Mtzr. Analysis of spontaneous
Rifr, Clar, and Cipr mutants
confirmed previous results indicating that mutations within the
rpoB gene, the 23S rRNA gene, and the
gyrA gene, respectively, are responsible;
also, several new mutant alleles were identified. Mtzr
mutants resulted most frequently, but not always,
from mutations in the rdxA gene. DNA fragments
containing each mutant allele could readily transform susceptible
H. pylori strains to resistance, confirming that
each mutant allele is responsible for the resistance phenotype.
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results and Discussion
References
subunit of RNA
polymerase (7). For metronidazole resistance,
although several different mechanisms may exist, the predominant
determinant has been shown to be the mutational inactivation of the
rdxA gene that encodes an oxygen-insensitive NADPH
nitroreductase (4, 9, 10, 13, 21).
MATERIALS AND METHODS
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Abstract
Introduction
Materials and Methods
Results and Discussion
References
4 in BHI-YE broth
(~105 cells/ml) and divided into 0.5-ml
aliquots. The number of aliquots was 12 to 30 (see Table 2). These
aliquots were allowed to grow for 3 days to obtain parallel,
independent cultures. The number of resistant mutants that emerged in
each culture was determined by plating the entire culture on BHI-YE
agar plates containing a selective antibiotic. The total number of
cells was determined by plating an appropriate
(105, 106, and
107) dilution of three cultures on nonselective
medium. Colonies on both selective and nonselective plates were counted
after incubation for 4 days. The frequency of resistant mutants was
expressed as the mean number of resistant cells divided by the total
number of viable cells per culture. For calculation of the mutation
rate, the most likely number of mutations per culture (m)
was first calculated from the distribution of numbers of resistant
mutants in the independent cultures by using an appropriate estimator (17). A number of different estimators (equations
developed by mathematicians) are available, each of which is valid for
a particular range of the m value. Then the mutation rate
(µ) per cell division was calculated as µ = m/Nt,
where Nt is the total cell number per culture (17).
TABLE 1.
Oligonucleotide primers
Selection of resistant mutants by the serial passage method. A serial passage technique as described by Haas et al. (5) was used to select Mtzr mutants. A 3-day-grown H. pylori strain was transferred onto agar plates containing one-half the MIC of metronidazole. After 3 days of incubation, the surviving cells were transferred onto the medium containing twice the prior selective concentration of the antibiotic. These plates were then incubated for 3 days. The process was repeated serially until no growth occurred or a predefined antimicrobial concentration was reached.
DNA manipulation. Chromosomal DNA from H. pylori strains was isolated as previously described (3). DNA sequencing was carried out using the thermocycling sequencing system with Thermo-Sequenase purchased from Amersham Life Sciences, Cleveland, Ohio. Other DNA manipulations including PCR and gel electrophoresis were performed by standard methods (18).
Natural transformation. Antibiotic-susceptible H. pylori strains were transformed using chromosomal DNA or a specific PCR fragment from the resistant strains by the method described previously (3). Briefly, recipient cells were heavily inoculated on cold BHI-YE agar plates and grown for 5 h, followed by addition of DNA (300 ng for chromosomal DNA or 30 ng for the PCR fragment) onto the bacterial lawn. After incubation for 20 h under microaerobic conditions the cells were streaked onto BHI-YE plates containing the selective antibiotic for selecting the transformants (the concentrations of antibiotics are the same as those used for selection of spontaneous mutations). A small aliquot of cells (after serial dilution) was plated on drug-free plates to determine the total cell number. After 4 days of incubation, transformants (single colonies) were obtained, and the transformation frequency was calculated as the fraction of the transformants compared with the total number of viable cells. In our experience, the amount of DNA used for transformation is at saturation.
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RESULTS AND DISCUSSION |
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Abstract
Introduction
Materials and Methods
Results and Discussion
References
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Frequencies of spontaneous mutations.
It is known
that there is huge variation in determining the frequency of
spontaneous mutants (average fraction of mutant bacteria in a few
replicate cultures). Fluctuation assays have been developed to
determine the mutation rate (probability of mutation event per cell per
generation). Following the methods described by Rosche and Foster
(17), we determined both the mutant frequency and mutation
rate of H. pylori strains that become resistant to
different antibiotics (Table 2). Based on
previous reports on the susceptibility of the majority of H. pylori clinical isolates, we choose the selective concentrations
of antibiotics as follows: 20 µg/ml for rifampin, 1 µg/ml for
ciprofloxacin, 0.5 µg/ml for clarithromycin, 8 µg/ml for
metronidazole, and 1 µg/ml for amoxicillin.
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and a mutation rate of ~1.5 × 108 were
observed for the three H. pylori strains tested (Table
2). This is a level comparable to that found to occur in
Escherichia coli (11, 17). Determination of the
frequency and rate of ciprofloxacin resistance
(Cipr) mutations in UA802 indicated that it is at
levels similar to those of Rifr.
We proceeded to examine the occurrence of mutations that confer
resistance to clarithromycin, metronidazole, and amoxicillin, the
antibiotics that are frequently used in triple therapy for H. pylori infection. No Clar
mutants were observed in 8 out of 12 independent cultures (strain UA802), whereas a few mutants were obtained in the other four cultures.
For metronidazole resistance, we had expected a relatively higher
frequency. However, no mutants were observed in 11 out of 15 independent cultures (strain 11639), while a few mutants were obtained
in the other 4 cultures. The calculated mutation rates for
Clar and for Mtzr were
<109 per cell division, which is about 20-fold
lower than that of Rifr. For amoxicillin
resistance, an extremely low frequency was observed: no single
resistant colony was obtained from 12 cultures of H. pylori 11639. As H. pylori strains exhibit high
genetic diversity, the mutant frequencies of
Clar, Mtzr, and
Amxr were examined using several other strains,
and similar results were observed (data not shown).
The low frequency of Clar is not surprising,
because mutations at only two particular bases (adenines at positions
2142 and 2143) in the 23S rRNA gene can confer resistance
(26). In addition, A-to-C and A-to-T mutants, although
conferring resistance, have defects in ribosomal function and cannot
compete with the wild type and A-to-G mutant for growth
(27). Moreover, the majority of H. pylori
Clar isolates contain mutations in both copies of
the 23S rRNA gene. Most possibly, this resulted from a spontaneous
mutation in one gene followed by gene conversion of the other copy by
homologous recombination. Thus, the observed mutation frequency may be
the product of the spontaneous mutation rate and the frequency of homologous recombination.
Although beta-lactams have been extensively used for treating
other infectious diseases, emergence of resistance to amoxicillin in H. pylori has been reported rarely
(1, 6, 23). Using the serial passage procedure in vitro,
no mutations that confer resistance to >0.25 µg of amoxicillin/ml
have been identified (5). Here we show that the frequency
of Amxr mutants is below the level that can be
determined in the present assay system
(
109). Currently, the mechanism of
amoxicillin resistance in H. pylori is not very clear,
although some reports suggested that mutation or modification of
penicillin binding proteins may be responsible (2, 12).
The extremely low frequency of Amxr (observed
both in vivo and in vitro) suggests that cooperative mutations in more
than one target may be required for Amxr in
H. pylori.
The low frequency of Mtzr determined in vitro is
in sharp contrast to the high incidence of Mtzr
in H. pylori clinical isolates as well as in the
H. pylori mouse model experiment (8). As
suggested by Martinez and Baquero (15), mutation
frequencies are probably much higher in the course of an infective
process than those determined in vitro, because bacteria growing in
vivo are frequently under environmental stress and challenge. In
addition, the actual concentration of metronidazole in vivo to which
the bacteria are exposed may not reflect the dose used in vitro. This
may account for part of the discrepancy between the observations in
vivo and in vitro, but this cannot explain the low frequency of
Mtzr compared to that of
Rifr, as both were determined in vitro under the
same conditions.
Given that the mutational inactivation of the rdxA gene is
the major determinant for Mtzr (4),
theoretically any mutation in the rdxA gene that leads to
the defect of RdxA enzyme renders H. pylori
Mtzr. Thus, a spontaneous mutation frequency
would be expected to be similar to or even higher than that of
Rifr. This prompted us to consider the unique
mechanism of metronidazole resistance. Wild-type H. pylori cells have a functional RdxA that reduces metronidazole
(nontoxic) to hydroxylamine (toxic), which is responsible for killing
the bacterium, whereas the rdxA mutant cannot reduce
metronidazole (4). We hypothesized that a few rdxA mutations had occurred but the mutant cells had been
killed due to the following possibilities. First, the hydroxylamine was produced from the wild-type cells and penetrated the mutant cells. In
other words, when only a very small fraction of rdxA mutants exist in a huge population of the wild-type cells, these mutant cells
cannot survive exposure to the drug, even though they themselves do not
reduce metronidazole to hydroxylamine. Second, within a short time
after the rdxA gene mutation occurs (in the same or the next
generation), the functional RdxA enzymes may remain in the cell and
produce hydroxylamine. In other words, the new rdxA mutants
may still have an Mtzs phenotype (phenotypic lag).
In a reconstruction experiment, we tested the first possibility using
an Mtzr 11639 mutant selected in the previous
experiment. The culture of these cells (~109
cells/ml) was diluted 107 and
108 so that there would be only a few
(1-10) cells in a 0.1 ml aliquot. These cells, either
alone or together with ~108 wild-type 11639 (Mtzs) cells, were plated on medium containing 8 µg of metronidazole/ml. After 3 days of incubation, a similar number
of colonies were observed for both conditions (i.e., with or without
Mtzs cells). As a control, plating
~108 wild-type cells alone did not give rise to
any Mtzr colonies. This result indicated that the
presence of a huge population of the wild-type cells does not affect
the survival of a few Mtzr mutants in the
metronidazole-containing medium. Currently, we suspect that the second
possibility, in which the rdxA mutation may not result
immediately in Mtzr (phenotypic lag), is most
likely responsible for the low frequency observed in vitro.
It was reported that Mtzr H. pylori can be readily selected out in vitro by serial passages on
increasing sublethal doses of metronidazole (5, 24). We
performed similar experiments to select Mtzr
mutants by the serial passage method. The MIC of metronidazole for both
strains 26695 and 11639 is 2 µg/ml. Consistent with previous results
(5, 24), we were successful in obtaining
Mtzr mutants from the both strains by the serial
passage method. The mutants can be obtained by one of the following
passage procedures: 1 
2 4 8 µg/ml, 1 2 2 8 µg/ml, 2 4 8 µg/ml, 2 2 8 µg/ml, or (the
simplest) 2 8 µg/ml.
Selection of resistant mutants by the serial passage procedure may be
more similar to the situation in vivo. At a particular site of
infection, there could exist a low concentration of metronidazole (metronidazole is usually used to treat parasitic and anaerobic bacterial infections) and thus a correspondingly low level of hydroxylamine, which may increase the mutation rate (hydroxylamine is a
DNA-damaging agent) but may not kill the bacteria. We observed that
pretreatment of H. pylori cells with 2 µg of
metronidazole/ml increased the frequency of Rifr
mutants about 10-fold (data not shown), which is in agreement with the
recent finding that metronidazole activation is mutagenic (20). Jenks et al. (8) determined the
proportion of the Mtzr mutants that emerged in
the mice originally infected with a single Mtzs
strain, and the results indicated that repeated exposure to sublethal doses of metronidazole in vivo encouraged the development of
resistance. Similarly, we observed the drastic increase in the
frequency of Mtzr mutants (resistant to 8 µg of
metronidazole/ml) in the cell population that was preincubated with 2 µg of metronidazole/ml (data not shown). Therefore, the mutagenic and
selective effects of the low doses of metronidazole account for the
high frequency of the Mtzr mutants in vivo as
well as in the serial passages in vitro.
Spectra of spontaneous mutations. (i) Rifampin resistance.
In
many bacteria, rifampin resistance is due to mutations in the
rpoB gene encoding the subunit of RNA
polymerase, and the Rifr mutations are
mainly located in a short region (200 to 300 bp) of the gene. We
proceeded to analyze a large number of Rifr
mutations for a detailed analysis similar to that done with E. coli (11, 19). We isolated 60 independent
Rifr mutants of strain UA802. The MIC of rifampin
for these mutants ranges from 32 to 256 µg/ml (Table
3), whereas the MIC for the wild-type
UA802 is 0.1 µg/ml. Of the 60 independent mutants analyzed, 57 were
identified to have single base substitutions at 14 different alleles of the rpoB gene (designated rpoB1 to
rpoB14) (Table 3). The nature of the other three mutants has
not yet been determined because we sequenced only the 330-bp fragment
of the rpoB gene (Table 1). For these three mutants, no
mutation was found in this region, and the mutations are probably in
another region of the rpoB gene. The sequence change of each
of the Rifr mutations within the rpoB
gene is shown in Fig. 1 and listed in
Table 3. Many of these spontaneous Rifr mutations
are identical to those identified previously by Heep et al.
(7). In addition, several new Rifr
alleles have been identified, including those encoding D530G, L525I,
I586P, S526L, H540R, and D530Y. All mutations identified were located
in a short region of the rpoB gene corresponding to cluster
I and cluster II of the Rifr-determining region
in E. coli (11, 19). The codon
corresponding to Asp530 was shown to be the most frequently mutated
site. Substitutions at amino acid residues 525, 527, 540, and 586 were
also identified multiple times. Four spontaneous
Rifr mutants were also isolated from strain
26695, which contained the rpoB2, rpoB3, or
rpoB4 alleles (Table 3). This confirms that Asp530 is the
most important rifampin-binding site.
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(ii) Clarithromycin resistance. Because of the low frequency of Clar mutations, only four independent Clar mutants were obtained and analyzed. The sequencing results showed that two of them are due to the mutation A2142G and the other two are due to A2143G (Table 3). This result is in agreement with the observations that these two types of mutation are predominantly associated with Clar in clinical isolates (22, 25).
(iii) Ciprofloxacin resistance. We analyzed 12 independent ciprofloxacin-resistant (Cipr) mutants of UA802, and four different alleles (base substitutions) of gyrA were found (Table 3). The GyrA protein contains a quinolone resistance-determining region (QRDR) (about 40 amino acids long) at the amino terminus. Mutations in this region of GyrA in many bacteria gave a high level of resistance to quinolones. There was a single report of ciprofloxacin-resistant H. pylori clinical isolates (16), in which several types of base substitutions leading to the amino acid changes in the QRDR of the GyrA were identified. The four Cipr alleles identified in our study all affect the codon Asp91 (changed to different amino acids) in the QRDR of the GyrA, indicating that this residue is the most important target site for ciprofloxacin binding. Two mutations, D91Y and D91N, are frequently found, and these were also identified by Moore et al. (16) in clinical isolates. The D91A mutation was not found in that study.
(iv) Metronidazole resistance. We obtained only four independent Mtzr mutants from strain 11639 by direct selection on 8 µg of metronidazole/ml. All of them were found to have a mutation in the rdxA gene (Table 3). One of them has a C-to-T mutation that leads to the amino acid change of Arg16 to Cys. A single base substitution mutation (C to T) was also found in another mutant that results in a change of Pro51 to Leu. The other two mutants were due to insertion of an adenine in a run of seven adenines, causing the shift of the reading frame (at position 64) that encounters a stop codon at position 73 (truncation of the protein).
Using the serial passage method, we were able to select many more Mtzr mutants. We analyzed the spectrum of the mutations in 18 independent mutants that were obtained from 18 independent passage experiments. An 850-bp DNA fragment covering the entire rdxA gene coding region as well as ~100 bp each of its upstream and downstream regions was sequenced (Fig. 2). In 2 of 18 mutants, no mutations were found; the sequence is identical to that of the wild type. The other 16 mutants contain mutations in the rdxA gene, which are shown in Fig. 2 and listed in Table 3. In total, 10 new rdxA alleles (rdxA4 to rdxA13) have been identified from this collection of Mtzr mutants. Mutations that occurred most frequently are frameshifts in simple nucleotide repeat sequences. The addition of an A in a run of seven A's (rdxA2) and the deletion of a T in a run of four T's (rdxA4) were observed five and two times, respectively. The deletion of an A in a run of three A's (rdxA8) and the addition of an A in a run of six A's (rdxA10) occurred once each. The other three insertion/deletion mutations that cause frameshifts are the addition of an A and a G (rdxA5), the deletion of a G (rdxA6), and the addition of 14 bp (rdxA12). The remaining four mutations are base substitutions, including three transitions (rdxA9, rdxA11, and rdxA13) and one transversion (rdxA7). Except for alleles encoding the frameshifts at simple nucleotide repeats, many rdxA alleles identified here are different from those identified in previous studies (4, 9, 10, 13, 21), indicating that these mutations occurred randomly in the rdxA gene and led to its inactivation.
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Transformation with mutant DNA.
The chromosomal DNAs
were isolated from all mutant (resistant) strains listed in Table
3, except for Clar mutants, which have been
studied previously (26). All the DNAs have the
ability to transform the susceptible strain to generate a
resistant isolate (data not shown). To test if the transformation ability is attributable to the specific mutant allele identified, DNA
fragments containing each mutant allele (i.e., PCR fragments of 360 bp
for gyrA, of 330 bp for rpoB, and of 850 bp for
rdxA) (Table 1) were used for transformation. Again, each
mutant allele (i.e., gyrA1 to gyrA4,
rdxA1 to rdxA13, and rpoB1 to
rpoB14) (Table 3) was able to readily transform the
susceptible parental strain to generate resistance to the corresponding
antibiotic. The transformation frequencies are in the range of
106 to 104
transformants per viable cell, which is at least several hundred-fold higher than the frequency of spontaneous mutation. As controls, the
recipient cells (total cell number was ~107 in
a typical transformation experiment) that received an aliquot of water
did not yield any resistant colonies. Therefore, each resistance
phenotype is indeed attributable to the corresponding mutant allele
identified. Transformation and recombination of the mutant allele from
the resistant cells to susceptible cells (of the same or other strains)
at a rate that is at least several hundred-fold higher than spontaneous
mutation frequency could contribute to the rapid spread of the mutant
allele in the bacterial population. Therefore, the emergence of
antibiotic resistance observed in vivo is probably due to the combined
effects of spontaneous mutation and recombination.
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ACKNOWLEDGMENTS |
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This work was supported in part by funding from the Canadian Bacterial Diseases Network (Centers of Excellence Program) to D.E.T., who is a Medical Scientist with the Alberta Heritage Foundation for Medical Research (AHFMR).
We thank Yong Leng for calculating the mutation rate.
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FOOTNOTES |
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* Corresponding author. Mailing address: Department of Medical Microbiology and Immunology, University of Alberta, Edmonton, Alberta, Canada T6G 2H7. Phone: (780) 492-4777. Fax: (780) 492-7521. E-mail: diane.taylor{at}ualberta.ca.
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Top
Abstract
Introduction
Materials and Methods
Results and Discussion
References
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Antimicrobial Agents and Chemotherapy, March 2001, p. 727-733, Vol. 45, No. 3
0066-4804/01/$04.00+0 DOI: 10.1128/AAC.45.3.727-733.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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