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Next Article 
Antimicrobial Agents and Chemotherapy, June 2001, p. 1607-1614, Vol. 45, No. 6
0066-4804/01/$04.00+0 DOI: 10.1128/AAC.45.6.1607-1614.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Effect of Drug Concentration on Emergence of
Macrolide Resistance in Mycobacterium avium
Kevin A.
Nash*
Childrens Hospital Los Angeles and University
of Southern California, Los Angeles, California
Received 22 September 2000/Returned for modification 28 December
2000/Accepted 1 March 2001
 |
ABSTRACT |
The emergence of antibiotic resistance in mycobacteria involves the
selection of mutant variants within a susceptible bacterial population.
However, it is unclear whether antimycobacterial drugs act just as
selective agents or can influence the rate of appearance of resistant
mutants. The present study was initiated to address this issue by
monitoring the effects of antimicrobial agents on the appearance and
growth of clarithromycin (CLR)-resistant (CLRr) bacilli in
broth cultures of Mycobacterium avium. Preexposure of
M. avium to CLR had a significant dose effect on the
emergence of resistance, with concentrations of 4 to 8 µg/ml
resulting in a maximal (~104-fold) increase in the number
of CLRr bacilli after a 4-day incubation. In addition, a
dose effect was found with azithromycin. The use of combinations of CLR
with either ethambutol (EMB) or rifabutin (RFB) resulted in fewer
resistant bacilli compared to the use of CLR alone. The lowest active
concentration of EMB (4 µg/ml) was equivalent to the EMB MIC (4 to 8 µg/ml) for the parental CLRs strain and the emergent
CLRr variants, and thus, the antiresistance effect was
probably the result of the bacteriostatic effect of EMB on
CLRr bacilli. However, RFB was an order of magnitude more
active (0.05 µg/ml) at reducing resistance than suggested by the MIC
of this agent (0.5 to 1 µg/ml). These results indicate that the
emergence of resistance was not simply the selection of a preexisting
subpopulation of resistant bacilli. Further analysis suggested that
early events in the emergence of resistance involved organisms
(progenitors) that acquired a resistance phenotype. In addition, the
progenitors appeared to be in a transient state, able to develop into a
stable resistant lineage in the presence of CLR, or able to revert to the wild type in nonselective conditions.
| |
INTRODUCTION |
Drug resistance is a difficult and
not uncommon problem in the treatment of mycobacterial diseases,
especially tuberculosis. To reduce the likelihood of resistance
emergence, patients with mycobacterioses are treated with multiple
antimycobacterial agents. Yet, despite the use of combination therapy,
new cases of secondary (i.e., emergent) drug resistance in
Mycobacterium tuberculosis are continually arising (P. M. Simone and S. W. Dooley,
http://www.cdc.gov/nchstp/tb/pubs/mdrtb/mdrtb.htm). Patient-associated determinants (e.g., compliance and immune state) are
important risk factors in the emergence of resistance (Simone and
Dooley, http://www.cdc.gov/nchstp/tb/pubs/mdrtb/mdrtb.htm); however, it is the genetic and metabolic states of the infecting microbial populations that ultimately determine if resistance will appear.
The focus of antimycobacterial treatment agents has been based, at
least initially, on susceptibility studies with wild-type and isolated
organisms expressing clinically significant levels of resistance. The
choice of agents used in drug combinations is prioritized further
largely on the basis of therapeutic efficacy. However, little is
understood of the processes involved in the acquisition of resistance,
and there may be agents that are relatively inactive as therapeutic
agents but that may have significant activity at preventing the
emergence of resistance. There is a precedent for this view, in that
treatment with the combination of clarithromycin (CLR) and ethambutol
(EMB) is effective at reducing the incidence of macrolide resistance
during the treatment of disseminated Mycobacterium avium
complex (MAC) disease (1). However, the addition of EMB has no significant effect on the reduction of total bacterial numbers.
Recently, Martinez and Baquero (10) reviewed the factors
that are known to influence the appearance of mutations associated with
increased levels of resistance to antimicrobial agents. Many of the
environmental factors that affect the appearance of resistant mutants
are clinically relevant, e.g., limiting nutrients, microbial competition, and antibiotic stress. The last factor is particularly interesting in that antibiotics can act as stressors (increasing mutation rates [10]) as well as be the selectors of any
resulting resistant mutants. Furthermore, the concentration of
antibiotic appears to influence the rate of drug resistance mutation
emergence (10).
The present study was initiated to investigate the effect of antibiotic
concentration and of antibiotic combinations on the emergence of
drug-resistant mutants in susceptible mycobacterial populations.
Furthermore, the study also questions whether the emergence of drug
resistance derives from a drug-resistant subpopulation that existed
prior to the addition of the antibiotic. To achieve these objectives, a
simple in vitro model system was used to monitor the emergence of
macrolide resistance in wild-type strains of MAC. Acquisition of
macrolide resistance in MAC is a useful phenotype for study as it is
based on a small number of possible mutations within the
peptidyltransferase region of the 23S rRNA gene (12, 13, 14,
15). Furthermore, a single base substitution in the
peptidyltransferase region is sufficient to confer high-level macrolide
resistance to mycobacteria (18) and the drug resistance phenotype of the mutants is independent of the specific mutation involved.
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MATERIALS AND METHODS |
Antimicrobial agents.
CLR (Abbott Laboratories, Abbott Park,
Ill.) was dissolved in methanol to give a maximum concentration of 6 mg/ml, which was then immediately diluted 1:2 with sterile 0.1 M
phosphate buffer (pH 6.8). Stock solutions of 10 mg of azithromycin
(AZM; Pfizer Inc., Groton, Conn.) and rifabutin (RFB; Pharmacia & Upjohn, Milano, Italy) per ml were prepared in methanol and ethanol,
respectively. EMB (Sigma Chemical Co., St. Louis, Mo.) was dissolved in
double-distilled water to 1 mg/ml and filter sterilized (pore size, 0.2 µm).
Media.
The media used for the culture of MAC were
Middlebrook 7H11 agar (Hardy Diagnostics, Santa Maria, Calif.) and a
modified Middlebrook 7H9 broth (7HSF broth) supplemented with 10%
oleic acid-albumin-dextrose-catalase (OADC; BBL, Becton Dickinson,
Cockeysville, Md.) and 1 g of Trypticase casein digest (BBL).
Antibiotic-containing agar plates were prepared with 7H11 agar base
(Difco, Detroit, Mich.) supplemented with 10% OADC and either 64 µg
of CLR per ml (7H11-CLR), 256 µg of AZM per ml (7H11-AZM), or 8 µg
of RFB per ml (7H11-RFB). All cultures were incubated at 37°C in room air.
Mycobacteria.
CLR-susceptible (CLRs) MAC strains
101 and 504 and CLR-resistant (CLRr) MAC strains 101R, 511, 512, and JJL004.2 are described elsewhere (14, 15).
Strains 101R and 511 are variants of strains 101 and 504 (14). Five CLRr variants of strain 101 (strains 101ER-1 through 101ER-5) and five CLRr variants of
strain 504 (strains 504ER1 through 504ER5) were selected on 7H11-CLR.
The strain 504 variants, 504c5 and 504c8, express an intermediate-level
CLR resistance (CLRi; CLR MIC, 16 to 32 µg/ml). These
variants were isolated from cultures of strain 504 grown on 7H11 agar
containing 16 µg of CLR per ml.
MIC determination and mutation analysis.
Susceptibility to
antimicrobial agents was assessed by a broth microdilution assay based
on previously described protocols (6). Briefly, drug
dilution series (at a 1.5× concentration) were prepared in 7HSF, and
0.1 ml was dispensed into the wells of 96-well, U-bottom microtiter
plates. Each well was inoculated with 7.5 × 104 CFU
of MAC in 0.05 ml of 7HSF broth. Bacterial growth was assessed visually
after the plates had been incubated for 5 days at 37°C. Previous
analysis confirmed that the microdilution-based assay was consistent
with a standard BACTEC radiometric, broth macrodilution assay
(7). Macrolide resistance in MAC is defined as a CLR MIC
of >32 µg/ml (although for CLRr MAC strains the CLR MIC
is >512 µg/ml) or an AZM MIC of >256 µg/ml. Previous studies have
confirmed that all CLRr MAC strains with 23S rRNA gene
mutations are also resistant to AZM (13, 14, 15).
The presence of the 23S rRNA gene mutation was detected by using a
modification of the Mismatch Detect II kit (Ambion, Inc., Austin, Tex.)
as described elsewhere (15).
In vitro model of the emergence of macrolide resistance.
Seed cultures of MAC strains were cultured in 7HSF broth until the
early stationary growth phase was reached (approximately 3 × 108 to 6 × 108 CFU/ml). Preliminary
experiments showed that the emergence of resistance in vitro was more
reproducible when seed cultures in the stationary growth phase were
used. The seed cultures were diluted 5- to 10-fold with fresh 7HSF
broth with or without the addition of antimicrobial agents (singly or
in combination), and the cultures were incubated for 4 days at 37°C.
During this period, samples (0.5 to 25 ml) were taken from the
cultures. The samples were sonicated for 10 min in a bath sonicator
(Gen-Probe, San Diego, Calif.) and then plated in triplicate onto 7H11
agar and 7H11-CLR to determine the total numbers of CFU per milliliter and the numbers of CLRr CFU per milliliter. Microscopic
examination of the cultures showed that sonication thoroughly dispersed
bacterial clumps. In selected experiments, the number of
macrolide-resistant CFU was determined by plating on 7H11-AZM as well
as 7H11-CLR.
For time course studies, 100- to 200-ml culture volumes were used;
however, in selected experiments, triplicate 40-ml culture volumes were
used in order to provide a sufficient sample size for statistical
analysis (Student's t test). Each experiment was performed
at least twice.
To characterize the heritable state of the emerging, resistant
organisms, seed cultures were diluted 10-fold and preincubated in the
absence of CLR for up to 10 h. Then CLR was added to the cultures at a
final concentration of 4 µg/ml and the incubation was continued for a
total of 4 days. Total numbers of CFU per milliliter and the numbers of
CLRr CFU per milliliter were determined as described above.
| |
RESULTS |
Does the concentration of CLR affect the emergence of macrolide
resistance?
Figure 1 shows the
effect of CLR concentrations of 8 and 32 µg/ml on the total numbers
of CFU per milliliter and the numbers of CLRr CFU per
milliliter over time in cultures of MAC strains 101 and 504. The number
of resistant bacilli (numbers of CLRr CFU per milliliter;
CLR MIC, >64 µg/ml) increased by more than 3 orders of magnitude
after 4 days of culture with 8 µg of CLR per ml. However, the numbers
of resistant bacilli in cultures containing 32 µg of CLR per ml were
15- to 30-fold less than the numbers in cultures containing 8 µg/ml.
Furthermore, the difference between the numbers of resistant bacilli in
cultures with 8 and 32 µg/ml was significant (P < 0.05) after only 2 days of incubation. Thus, the increase in the
numbers of resistant bacilli was dependent on the concentration of CLR.
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FIG. 1.
Effect of CLR ( , 8 µg/ml;  , 32 µg/ml) on the
total numbers of CFU per milliliter and the numbers of CLRr
CFU per milliliter in cultures of MAC strains 101 and 504 over a 4-day
incubation. Conditions that resulted in significantly lower numbers of
CLRr organisms compared to the numbers in cultures
containing 8 µg of CLR per ml are indicated (*, P < 0.05; **, P < 0.001). For clarity, error bars
are not shown, as they are all within the datum point symbols.
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Clumping is often a confounding factor in relating the numbers of CFU
to the number of mycobacterial bacilli. However, the results shown in
Fig. 1 are unlikely to be a consequence of clumping, as sonication of
the cultures prior to plating thoroughly dispersed bacterial
aggregates. Furthermore, CLR had no effect on bacterial aggregates in
cultures of CLRr MAC strains. Thus, clumping of any
preexisting CLRr bacilli would be expected to be the same
in the cultures containing CLR at either 8 or 32 µg/ml.
A decrease in the numbers of total viable organisms was expected, as
the CLR concentrations were above the MICs for both strains (Table
1). In contrast to the numbers of
CLRr CFU, however, the changes in the total numbers of CFU
were relatively independent of the CLR concentration.
In order to expand on the results shown in Fig. 1, the effect of a
wider range of CLR concentrations on the emergence of resistance was
investigated (Fig. 2). As before, there
was a dose effect of CLR on the numbers of resistant organisms present
after 4 days of culture. Incubation in 4 µg of CLR per ml resulted in
the greatest increase (approximately 10,000-fold) in the number of
CLRr organisms, with significantly fewer resistant
organisms emerging at CLR concentrations of 
2 and 16 µg/ml. For MAC
strain 101, 4 and 8 µg of CLR per ml tended to result in equivalent
numbers of CLRr organisms. Interestingly, the peak CLR
concentration for the emergence of resistance is the same as the CLR
MICs for MAC strains 101 and 504 and at least 2 orders of magnitude
below the CLR MICs for CLRr MAC strains (Table 1). The
lower number of CLRr variants in the cultures with 1 µg
of CLR per ml was not a result of out-competition by the more abundant
CLRs organisms, since the total population density remained
relatively low (<2 × 107 CFU/ml) during the 4-day
incubation.
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FIG. 2.
Total numbers of CFU per milliliter () and numbers of
CLRr CFU per milliliter (bars) after 4 days of culture in
the presence of CLR concentrations of 1 to 16 µg/ml. For each strain,
conditions that resulted in significantly lower numbers of
CLRr organisms compared to the numbers in cultures
containing 4 µg of CLR per ml are indicated (*, P < 0.05; **, P < 0.01).
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Similar to the results obtained with CLR, AZM showed a dose-dependent
effect on the numbers of macrolide-resistant organisms (Fig.
3). Furthermore, the number of resistant
organisms obtained by plating on 7H11-AZM was not significantly changed
compared to the number obtained by plating on 7H11-CLR, confirming that CLR and AZM were selecting the same population of resistant organisms. The peak concentrations of AZM used were 256 µg/ml for strain 101 and
128 to 256 µg/ml for strain 504. However, unlike CLR, these
concentrations were higher than the AZM MIC, i.e., 32 µg/ml for
strain 101 and 16 µg/ml for strain 504. The phenotype of slightly greater susceptibility for strain 504 may explain the less distinct peak in the number of resistant bacilli and the greater bactericidal activity of AZM against this strain.
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FIG. 3.
Total numbers of CFU per milliliter () and numbers of
CLRr CFU per milliliter (bars) after 4 days of culture in
the presence of AZM (32 to 512 µg/ml) and CLR (4 and 64 µg/ml). For
each strain, conditions that resulted in significantly lower numbers of
CLRr organisms compared to the numbers in cultures
containing 4 µg of CLR per ml are indicated (*, P < 0.05; **, P < 0.01).
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Although the experiments represented by Fig. 1 to 3 were reproducible,
the results were dependent on harvesting of the seed cultures in the
stationary phase. Seed cultures that were in the early to the
mid-exponential phase generated few resistant bacilli by day 4 in the
experimental system. Furthermore, the resistant organisms that were
present at day 4 tended to be unevenly distributed between experimental
cultures, leading to so-called jackpot cultures.
Characterizing the emergent CLRr bacilli.
In order
to confirm the mutational basis of the macrolide resistance, the DNAs
isolated from CLRr variants were analyzed for 23S rRNA gene
mutations. For each MAC strain, this analysis was applied to five
CLRr variants derived from the seed cultures at time zero
and 25 CLRr variants isolated from broth cultures on day 4. All 60 variants were found to have mutations in the peptidyltransferase
region of the 23S rRNA gene, consistent with the macrolide resistance phenotype (data not shown). Although the specific mutations were not
confirmed by DNA sequencing, the banding patterns generated by the
mismatch assay did not indicate a shift in the base substitutions between the two time points.
Despite the mutation uniformity, there was a clear difference in the
rate of colony appearance on the experimental 7H11-CLR plates
inoculated with the seed cultures compared to that on the plates
inoculated with the broth cultures from day 4. The agar plates
inoculated with seed cultures required an incubation of ~10 days
before the first colonies appeared. Furthermore, the numbers of visible
colonies increased over the following 4 to 5 days. In contrast,
colonies were visible after an incubation of only 5 to 6 days for the
agar plates inoculated with the experimental broth cultures from day 4, and all the colonies appeared concurrently. The 5- to 6-day incubation
period was consistent with the growth of CLRr MAC strains
(e.g., strains 101R and 511) plated on 7H11-CLR.
These observations suggest that the source organisms for the
macrolide-resistant colonies derived from the seed cultures differed from the source organisms derived from the CLR-exposed broth cultures.
Modeling a preexisting macrolide-resistant subpopulation.
The
low optimal concentration of CLR (4 µg/ml) for the emergence of
resistance suggests that resistance does not derive from a highly
resistant preexisting subpopulation. To confirm this, the emergence of
resistance from a highly resistant preexisting subpopulation was
modeled by using a collection of CLRr MAC strains (Table
2). Four of the strains emerged in vivo
during treatment of disseminated disease with macrolide therapy
(strains 101R, JJL004.2, 511, and 512), and the remaining two strains
were selected in vitro (strains 504ER1 and 504ER2). The 23S rRNA genes of these strains were analyzed by either DNA sequencing
(14) or mismatch assay (strains 504ER1 and 504ER2), and
all strains were confirmed to have a mutation consistent a macrolide
resistance phenotype. Furthermore, all strains expressed the resistance
phenotype constitutively; i.e., resistance was not affected by
preincubation in macrolide (data not shown).
Table 2 shows the effect of CLR on the generation times of the six
CLRr strains. Thus, growth was not significantly affected
(P value range, 0.09 to 0.96) by the presence of either 4 or
64 µg of CLR per ml, irrespective of the origins of the resistant
strains. Similarly, AZM (tested up to 256 µg/ml) had no significant
effect on the growth rates of macrolide-resistant bacilli (data not
shown). The growth rates of all the CLRr strains were
significantly lower than those of CLRs strains 101 (P < 0.002) and 504 (P < 0.05).
In order to determine if the growth of CLRr bacilli was
affected by an excess of CLRs bacilli, isogenous strains
504 (CLRs) and 511 (CLRr) were mixed at a ratio
of 106:1. As before, the growth rate of the
CLRr strain was unaffected by the presence of CLR (Table
2).
Thus, if the emergence of resistance from a wild-type bacterial
population was simply the result of clonal expansion of preexisting CLRr bacilli, then the concentration of CLR should have had
little effect on the increase in numbers of resistant organisms.
Drug combinations and the emergence of resistance.
The results
described above indicate that the emergence of resistance is affected
by the concentration of macrolides in a way not predicted by the
susceptibility profiles of either the susceptible or the resistant
organisms (Table 1). This raises the possibility that susceptibility
profiles may not predict how drug combinations affect the emergence of
macrolide resistance. To investigate this, the emergence of macrolide
resistance was compared between cultures containing CLR alone (4 µg/ml) and cultures containing CLR in combination with either EMB or
RFB (Fig. 4).
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FIG. 4.
Total numbers of CFU per milliliter () and numbers of
CLRr CFU per milliliter (bars) after 4 days of culture in
the presence of CLR (4 µg/ml) alone or CLR (4 µg/ml) in combination
with either EMB (B, 1 µg/ml; C, 2 µg/ml; D, 4 µg/ml; E, 8 µg/ml) or RBT (F, 0.0125 µg/ml; G, 0.025 µg/ml; H, 0.05 µg/ml;
I, 0.1 µg/ml). Conditions that resulted in significantly lower
numbers of CLRr organisms compared to the numbers in
cultures containing CLR alone are indicated (*, P < 0.05).
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The addition of EMB at concentrations 
4 µg/ml resulted in
significantly fewer resistant bacilli for both strains. Thus, the lowest concentration of EMB that significantly reduced the emergence of
resistance was equivalent to the MIC of this agent for strains 101 and
504 and their derived CLRr variants (Table 1). This
indicates that the effect of EMB was probably the result of
bacteriostasis of the CLRr (and CLRs) bacilli.
The use of combinations of CLR with 0.05 µg of RFB per ml showed a
significantly reduced rate of emergence of CLR resistance compared to
that from the use of CLR alone (Fig. 4). In contrast to the effect of
EMB, 0.05 µg of RFB per ml was 10- to 20-fold lower than the RFB MIC
determined for the parental strains, 101 and 504, and the
CLRr variants (Table 1). In fact, the level of growth of
resistant organisms in 0.05 µg of RFB per ml, with or without 4 µg
of CLR per ml, was not discernibly different from the level of growth in medium alone (data not shown). This indicates that the effect of RFB
on the emergence of CLR resistance was not the result of inhibition of
the growth of the CLRr bacilli by RFB.
Despite the significant effect on the emergence of resistance, the
addition of EMB (up to 8 µg/ml) or RFB (up to 0.1 µg/ml) only
marginally affected the bactericidal activity of CLR (Fig. 4).
Clonal expansion versus "nascent" resistance.
If the
emergence of resistance does not derive from a preexisting resistant
population, then there must be, at least initially, a period of nascent
resistance (i.e., a period when organisms are acquiring a resistance
phenotype). Furthermore, it must be this acquisition period that is
dependent on the CLR concentration. Once high-level phenotypic
resistance has been acquired, the numbers of resistant bacilli will
increase by cell division (i.e., clonal expansion of the
CLRr bacilli), which is unaffected by the CLR concentration
(Table 2).
The time point when the emergence of resistance is predominantly the
result of clonal expansion of CLRr bacilli can be assessed
by determining when changes in number of CLRr bacilli are
refractory to high concentrations of CLR (64 µg/ml). To determine
this point, replicate 100-ml cultures of MAC 101 (7.6 × 107 CFU/ml) were set up with an initial CLR concentration
of 8 µg/ml. At time zero and daily thereafter, a different replicate
culture was supplemented with CLR to a final concentration of 64 µg/ml. In addition, samples were taken daily from all cultures for
determination of the total numbers of CFU per milliliter and the
numbers of CLRr CFU per milliliter.
The number of CLRr bacilli was equivalent in all cultures
on day 1 and day 2 (Fig. 5). By day 4, however, the cultures maintained with 8 µg of CLR per ml for at least
3 days had significantly (P < 0.01) more
CLRr bacilli than the other cultures, i.e., 2,375 ± 650 versus 55 ± 21 CFU/ml, respectively. The total numbers of CFU
per milliliter were equivalent in all the cultures (data not shown).
Strain 504 was analyzed in a similar manner, using a shift in the CLR
concentration from 4 to 64 µg/ml. Cultures maintained with 4 µg CLR
per ml for at least 2 days resulted in significantly (P < 0.01) higher numbers of CLRr CFU than cultures in
which the CLR concentration was increased prior to day 2, i.e.,
977 ± 294 versus 30 ± 14 CFU/ml, respectively.
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FIG. 5.
Effects of increasing concentrations of CLR on the
numbers of CLRr organisms in cultures of MAC strain 101. Replicate cultures were initiated with 8 µg of CLR per ml; and on day
0 ( ), day 2 (), day 3 ( ), or day 4 () different cultures
were supplemented with CLR to final concentration of 64 µg/ml. For
clarity, the results for the culture supplemented on day 1 are not
shown. Error bars are not shown, as they are all within the datum point
symbols.
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Thus, clonal expansion appeared to become the predominant cause of the
increase in the CLRr population after a 2- to 3-day
exposure to concentrations of CLR optimal for the emergence of resistance.
Hypermutable organisms, intermediate-level resistance, and
the emergence of resistance.
If the acquisition of
resistance is a consequence of a stable hypermutable (mutator) state,
then the probability of mutation should be higher for the emergent
CLRr variants than for the parental strains. To investigate
this, strain 101, 4 variants selected in vitro, and a pool representing 1,000 variants selected in vitro were each plated on 7H11 and 7H11-RFB,
and the frequency of RFB-resistant (RFBr) variants was
determined. The frequencies of RFBr bacilli of strain 101 and the mean frequencies for the individual CLRr variants
and the variant pool were 6.2 × 10
9 ± 1.5 × 109, 4.7 × 109 ± 2.5 × 109, and 3.4 × 109 ± 2.7 × 109 respectively. Therefore, the probability of
acquiring resistance to RFB for the CLRr variants was not
greater than that for the parental strain.
An alternative explanation for the effect of CLR on the emergence of
resistance is that CLR causes a transient increase in the general
mutation rate in a dose-dependent manner. CLR could then select the
CLRr organisms from the increased pool of variants. To
explore this possibility, the frequency of resistance to RFB was
determined in MAC cultures exposed to CLR (4 µg/ml) for 2 days. This
period was long enough for a detectable increase in CLRr
variants, without too severely depleting the total bacterial population
(Fig. 1). In the experiment the numbers of viable organisms decreased
from 7.45 × 107 to 6.09 × 107
CFU/ml, whereas the numbers of CLRr organisms increased
from 0.7 to 6.8 CFU/ml; this represents a frequency shift of 9.4 × 109 to 1.1 × 107.
The frequency of RFBr bacilli in the strain 101 seed
cultures was 7.9 × 109 ± 1.9 × 109, whereas it was 3.1 × 109 ± 1.3 × 109 for cultures incubated in 4 µg of CLR
per ml for 2 days. Thus, incubation of MAC with CLR was not associated
with an increase in the appearance of other (i.e.,
non-CLRr) variants. These results suggest that the
progenitors of CLR resistance were drug specific.
It is possible that the appearance of CLRr bacilli is a
multistep process, with organisms initially gaining an
intermediate-level resistance phenotype (CLRi). If an
intermediate phenotype is a step in the emergence of CLRr,
then CLRi bacilli (CLR MIC, 16 to 32 µg/ml) should be
more likely than wild-type bacilli to mutate to high-level resistance.
To investigate this, the relative mutability of wild-type strain 504 and the CLRi derivatives, 504c5 and 504c8, was assessed by
plating samples of each strain (1010 CFU) on 7H11-CLR. The
frequencies of the derived numbers of CLRr CFU (relative to
the total numbers of CFU plated) for strains 504, 504c5, and 504c8 were
7.0 × 109 ± 2.6 × 109,
4.4 × 109 ± 0.6 × 109,
and 8.1 × 109 ± 0.8 × 109. Thus, strains with the wild-type and
CLRi phenotypes were equally likely to mutate to high-level
resistance. This suggests that an CLRi phenotype per se is
not a required step in the acquisition of high-level resistance.
Characterization of the CLRr progenitors.
If the
initial period of resistance acquisition is the consequence of
phenotypic lag, then the mutated state (i.e., base substitution within
the 23S rRNA gene) of the source organisms should be heritable. To
investigate this, the heritable state of the resistance progenitors was
determined by preincubation of the cultures at relatively low bacterial
density (~5 × 107 CFU/ml) for up to 10 h
before the addition of CLR. This preincubation step placed the seed
organisms (in the early-stationary-growth phase) in conditions suitable
for exponential growth. The results of a representative experiment are
shown in Fig. 6.
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FIG. 6.
Effects of delaying the addition of CLR (4 µg/ml) on
the emergence of CLR resistance in MAC strain 101. Seed cultures were
diluted 10-fold with fresh medium and incubated from 0, 3, 6, and
9 h before the addition of CLR (4 µg/ml). The total numbers of
CFU per milliliter and the numbers of CLRr CFU per
milliliter were determined after a total of 4 days in culture. For
clarity, error bars are not shown, as they are all within the datum
point symbols.
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In repeated experiments, preincubation of the cultures without CLR for
approximately one generation (~9 h) reduced the numbers of
CLRr organisms by 5- to 10-fold, although in one case the
reduction was 40-fold (data not shown). Delay of the addition of CLR
resulted in a decrease in the numbers of emergent CLR bacilli. Thus,
the phenotype of the resistant progenitors was not committed or
heritable. Furthermore, the evidence suggests that the progenitors
existed prior to the addition of CLR.
| |
DISCUSSION |
Study of the emergence of resistance to macrolides in mycobacteria
has several advantages over the study of other resistance phenotypes.
First, relatively few mutations are associated with macrolide
resistance in mycobacteria, and all are in the peptidyltransferase region of the 23S rRNA gene (12, 13, 14, 15). Second, all
resistant mutants have the same phenotype; i.e., the CLR MIC is high
(>64 µg/ml [14, 15]) and the growth rates are
refractory to the presence of macrolides (up to at least 64 µg/ml for
CLR). However, the acquisition of a 23S rRNA gene mutation does confer a fitness disadvantage in the absence of macrolide, shown by the reduced rates of growth of macrolide-resistant M. avium
relative to those for the wild type (Table 2). This effect has been
reported previously for both mycobacteria (K. A. Nash, Program
37th Intersci. Conf. Antimicrob. Agents Chemother. abstr. S-84, 1987)
and some clarithromycin-resistant Helicobacter pylori
mutants (3, 22).
The effect of the drug concentration on the emergence of other types of
resistance, such as quinolone resistance (4), is complicated by the fact that the phenotype (i.e., the level of resistance) can vary with the underlying mutation. This variability results in the selection of decreasing numbers of mutants as the concentration of antibiotic increases (4). This
association between antibiotic concentration and the selection of
different mutant populations has been modeled by Martinez and Baquero
(10). However, the results presented here suggest that the
emergence of macrolide resistance is more complex than just the
selection of resistant populations.
Macrolides have a dose-dependent effect on the emergence of specific
resistance in M. avium. The maximal increase in the numbers of resistant bacilli was at 4 µg of CLR per ml, which is the same as
the CLR MIC for the susceptible parental M. avium strains. The use of combinations of CLR and either EMB or RFB resulted in fewer
resistant organisms than the use of CLR alone. For EMB, this effect
could be explained by its bacteriostatic effect on the resistant
bacilli. However, the effect of RFB is not explained by a direct effect
of RFB on resistant organisms. Thus, the emergence of
macrolide-resistant M. avium is inconsistent with the
hypothesis that drug resistance emerges predominantly from a
preexisting resistant subpopulation.
The results presented here do not rule out the existence of preexisting
resistant subpopulations, just that they have a minor role in the
initial stages of the emergence of CLR resistance. In addition, the
results of the present study are not contradictory to the role of
random mutation, in the absence of selective pressure, as an important
process in bacterial evolution. However, CLR-resistant M. avium appears to derive from a specific population of organisms that act as the progenitors of resistance. Furthermore, these progenitors appear to be in an unstable or transient state, i.e., the
bacilli do not appear to be committed to the acquisition of resistance,
although they are specific for the acquisition of CLR resistance. The
progenitors appear to be more abundant in stationary-phase cultures.
This is consistent with the increased mutability of other bacterial
species, including Mycobacterium smegmatis, when they are in
the stationary phase or under starvation conditions (8, 10,
21).
An important step in the progression of the progenitor to a resistant
bacillus is the addition of the antimicrobial agent. Consequently, the
antimicrobial agent probably has two effects, first, as an
environmental stressor that affects the mutability of the progenitors
and, second, as a selective agent for the bacilli that acquire a
mutation that confers resistance. Since the only organisms that will be
released from the inhibition by the antimicrobial agent are those that
acquire the appropriate mutation, the combination of the two effects
may explain the apparent drug specificity of the progenitors.
Furthermore, since the mutant bacilli need to replace ~50% of their
ribosomes to acquire a high-level macrolide resistance phenotype
(19), phenotypic lag probably accounts for a large
proportion of the delay between the addition of CLR and the appearance
of highly resistant bacilli.
Superficially, the results of the present study appear to support the
directed mutation hypothesis (2). However, this is misleading since the most important step in the generation of the
progenitors (i.e., acquisition of the uncommitted state) happens in the
absence of CLR, and thus, CLR does not truly direct the evolution of
the resistant variants. In addition, evidence presented by others has
largely discounted the directed mutation hypothesis (5, 9, 16,
21). Previous reports proposed that hypermutable variants or
mutators aid bacterial evolution (17, 20). The evidence
presented here suggests that stable mutators do not play a significant
role in the emergence of CLR resistance in MAC. However, the results
are consistent with the presence of a population of organisms in a
transient hypermutable state (17). In Escherichia coli, transient hypermutable (or adaptive mutation) is dependent on expression of the homologous recombination proteins RecA and RecBCD
(17) and is regulated by the SOS response
(11).
Understanding the mechanisms of the emergence of drug resistance and
the factors that affect it will aid in the design of improved
preventive strategies. This will occur by several means. Experimental
data will help refine the mathematical models of the emergence of
resistance and also provide a basis for predicting the probability of
the development of resistance to new agents (10).
Furthermore, the results of the drug combination experiments suggest
that susceptibility results may not be a good indicator of the utility
of antimicrobials for prevention of the emergence of resistance to
other agents. Thus, drugs that are not considered highly active may
still have a role in reducing or preventing resistance. The screening
of antibiotics for the ability to reduce the level of resistance has
been proposed by others (4, 10), and the results presented
here lend support to that approach. However, the problem may be how to
assess the emergence of resistance in a straightforward, reproducible,
and clinically relevant manner (10).
| |
ACKNOWLEDGMENTS |
I thank Clark B. Inderlied for the use of laboratory resources
and for comments in preparation of the manuscript and Priscilla A. Aralar for technical assistance.
This work was funded in part by NIH contract NO1-AI-25140.
| |
FOOTNOTES |
*
Mailing address: Department of Pathology and Laboratory
Medicine, Childrens Hospital Los Angeles, 4650 Sunset Blvd., Mailstop 103, Los Angeles, CA 90027. Phone: (323) 669-5670. Fax: (323) 671-3871. E-mail: kanash{at}usc.edu.
| |
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Antimicrobial Agents and Chemotherapy, June 2001, p. 1607-1614, Vol. 45, No. 6
0066-4804/01/$04.00+0 DOI: 10.1128/AAC.45.6.1607-1614.2001
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Top
Abstract
Introduction
