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Antimicrobial Agents and Chemotherapy, November 1999, p. 2600-2606, Vol. 43, No. 11
0066-4804/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Drug Tolerance in Mycobacterium
tuberculosis
Robert S.
Wallis,1,*
Shripad
Patil,2
Seon-Hee
Cheon,1,3
Kay
Edmonds,1
Manijeh
Phillips,1
Mark D.
Perkins,4,5,
Moses
Joloba,6
Alice
Namale,6
John L.
Johnson,1
Lucileia
Teixeira,4
Reynaldo
Dietze,4
Salman
Siddiqi,7
Roy D.
Mugerwa,6
Kathleen
Eisenach,8 and
Jerrold J.
Ellner1
Case Western Reserve University, Cleveland
Ohio1; National Institute of Mental
Health and Neuroscience, Bangalore, India2;
Ewha Womans University Hospital, Seoul, Republic of
Korea3; Federal do Espírito
Santo, Vitória, Brazil4; Duke
University Medical Center, Durham, North
Carolina5; Makerere University, Kampala,
Uganda6; Becton Dickinson, Sparks,
Maryland7; and University of Arkansas
Medical Center, Little Rock, Arkansas8
Received 1 June 1999/Returned for modification 3 August
1999/Accepted 13 August 1999
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ABSTRACT |
Although Mycobacterium tuberculosis is eradicated
rapidly during therapy in some patients with pulmonary tuberculosis, it can persist for many months in others. This study examined the relationship between mycobacterial drug tolerance (delayed killing in
vitro), persistence, and relapse. It was performed with 39 fully
drug-susceptible isolates from a prospective trial of standard short-course antituberculous therapy with sputum smear-positive, human
immunodeficiency virus-uninfected subjects with pulmonary tuberculosis
in Brazil and Uganda. The rate of killing in vitro was determined by
monitoring the growth index (GI) in BACTEC 12B medium after addition of
drug to established cultures and was measured as the number of days
required for 99% sterilization. Drugs differed significantly in
bactericidal activity, in the following order from greatest to least,
rifampin > isoniazid-ethambutol > ethambutol
(P < 0.001). Isolates from subjects who had relapses (n = 2) or in whom persistence was prolonged
(n = 1) were significantly more tolerant of
isoniazid-ethambutol and rifampin than isolates from other subjects
(P < 0.01). More generally, the duration of persistence during therapy was predicted by strain tolerance to isoniazid and rifampin (P = 0.012 and 0.026, respectively). Tolerance to isoniazid-ethambutol and tolerance to
rifampin were highly correlated (P < 0.001). Tolerant
isolates did not differ from others with respect to the MIC of
isoniazid; the rate of killing of a tolerant isolate by
isoniazid-ethambutol was not increased at higher drug concentrations.
These observations suggest that tolerance may not be due to
drug-specific mechanisms. Tolerance was of the phenotypic type,
although increased tolerance appeared to emerge after prolonged drug
exposure in vivo. This study suggests that drug tolerance may be an
important determinant of the outcome of therapy for tuberculosis.
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INTRODUCTION |
There is substantial variability in
the response to therapy of pulmonary tuberculosis (TB), even in those
patients infected with fully drug-sensitive isolates. In some patients,
Mycobacterium tuberculosis is rapidly eradicated and
patients are cured after as little as 2 to 3 months of ultra-short-term
chemotherapy (2-4, 14). Yet, in others, viable organisms
persist in sputum for many months, despite drug susceptibility in
vitro. The risk of relapse is increased in individuals in whom sputum
sterilization is delayed (1, 8, 17). The factors responsible
for mycobacterial persistence (prolonged culture positivity during
therapy) are not well understood but may include mycobacterial and host
biological factors, as well as host behavioral factors.
Studies of tolerance, the ability to evade the bactericidal activity of
antimicrobial drugs, may be relevant to these observations. The
hallmark of tolerance is bacteriostasis with reduced or delayed bactericidal activity (for a review, see reference
19). Tolerance in streptococci can be induced by
selective depletion of the medium of certain nutrients, exposure to
certain drugs, or more generally, existence in the stationary growth
phase. Tolerance may be an important factor that determines the risk of
relapse in patients with streptococcal endocarditis (13).
Mycobacterial persistence and relapse may in part be determined by drug
tolerance. Like the streptococcus, M. tuberculosis resists
killing when it is in the stationary growth phase. The sterilizing
activity of isoniazid (INH), for example, is reduced nearly 1,000-fold
in stationary-phase cultures (12). Sterilizing activity may
be essential for prevention of relapse of TB, yet the relationship of
mycobacterial drug tolerance to treatment outcome has not been studied.
Many clinical laboratories use the BACTEC TB 460 system (Becton
Dickinson, Sparks, Md.) for rapid detection and susceptibility testing
of mycobacteria. It is based on detection of
14CO2, produced by oxidation of
[14C]palmitic acid. In the study described in this
report, a simple method that used the BACTEC system to measure the rate
of killing of M. tuberculosis was developed. The method was
used to examine the relationship between tolerance in vitro and
persistence in vivo during standard therapy for TB.
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MATERIALS AND METHODS |
Mycobacterial isolates and clinical data were obtained from a
prospective study conducted in Uganda and Brazil and reported previously (21). Briefly, patients with initial episodes of sputum smear-positive pulmonary TB who had not received prior therapy
were prospectively recruited at TB control clinics in Kampala, Uganda,
and Vitória, Brazil. All patients gave informed written consent
for human immunodeficiency virus testing and study participation. The
study protocol was approved by the institutional review boards of Case
Western Reserve University (Cleveland, Ohio), Makerere University
(Kampala, Uganda), Universidade Federal do Espírito Santo
(Vitória, Brazil), Duke University (Durham, N.C.), and the
University of Arkansas (Little Rock). Tuberculosis was presumptively
diagnosed by a positive acid-fast smear of sputum and a compatible
chest radiograph and was subsequently confirmed by culture. Serology
for human immunodeficiency virus type 1 was performed for all subjects;
seropositive individuals were excluded from the study. Subjects were
also subsequently excluded from this analysis if their initial isolates
were resistant to INH, rifampin (RIF), pyrazinamide, or ethambutol
(EMB) or the duration of follow-up was less than 180 days.
Subjects were treated with INH, RIF, EMB, and pyrazinamide daily at
standard doses for 2 months, followed by daily treatment with INH and
RIF for 4 months. Patients were evaluated on days 0, 2, 4, 7, 14, and
30, monthly until therapy was completed, and then bimonthly. At each
evaluation, a medical history was taken and a physical examination was
performed. Multiple sputum specimens were obtained. These were
processed and analyzed as described below. Patients were hospitalized
for the initial 2 weeks of treatment, after which time therapy was
self-administered. Compliance was assessed at each clinic visit by
review of dispensing records, clinic attendance, and urinary isoniazid
metabolite testing (MycoDyn Uritec; DynaGen, Inc., Cambridge, Mass.).
Subjects were classified as treatment failures if M. tuberculosis was repeatedly isolated from sputum on days 120 to
180 in conjunction with symptoms and radiographic findings consistent
with active tuberculosis.
Sputum processing and culture.
Specimens were digested with
N-acetyl cysteine and were then decontaminated by addition
of an equal volume of 4% NaOH-2.9% sodium citrate, as described
previously (21). The specimen was then cultured on
Middlebrook Cohn 7H10 and 7H10S (antibiotic containing) agar medium and
in BACTEC 12B liquid medium. Decontamination of sputum with NaOH has
been shown to reduce the numbers of CFU by 80% (23).
However, this approach was adopted because use of NaOH resulted in
reduced contamination rates compared to those from the use of
antibiotic-containing medium and because the reduction in CFU was
consistent among specimens in a pilot study. Sputum cultures were
performed on site in Kampala and Vitória. Growth indices (GIs)
were measured daily after inoculation into BACTEC 12B medium. The day
of collection of the last sputum specimen to indicate growth in BACTEC
12B medium (GI 
10) within 20 days of inoculation (rapid growth)
was determined retrospectively for each subject. Specimens that were
contaminated with organisms other than M. tuberculosis were
excluded from analysis.
Measurement of drug tolerance.
Isolates were shipped to Case
Western Reserve University for studies of tolerance. Isolates were
placed into BACTEC 12B medium (Becton Dickinson) and were maintained in
culture at 37°C without agitation. GIs were monitored daily until a
value of 250 was reached, at which time 100-µl samples were removed
for inoculation of four replicate bottles. These were cultured until
the GI again reached 250, at which time EMB (final concentration, 2.5 µg/ml), is INH (0.1 µg/ml), or RIF (2.0 µg/ml) was added. Control
cultures were maintained without drug. The GI was monitored daily. The
mean coefficient of variation of GIs for replicate cultures on the day
of drug addition (day 0) was 8.3%. The mean day 0 GI among all
isolates was 277, with a coefficient of variation of 17.7%. To adjust
for these minor differences in baseline values, GIs for each culture
bottle in studies of clinical isolates were reported as GI × 250/day 0 GI.
Susceptibility and MIC testing.
Aliquots of 100 µl were
removed from cultures in BACTEC 12B medium when the GI reached 250. These were placed into culture in fresh bottles to which drug had been
added. Control cultures (without drug) were inoculated with an aliquot
that had been diluted 1:100 in bovine serum albumin 2 mg/ml and Tween
80 (0.02%). The GI was monitored daily until the control cultures
reached a GI of 30, at which time the increment in GI over the
subsequent 24 h was recorded. The MIC was reported as the lowest
concentration of drug resulting in an increment of GI less than that of
the control culture.
Data analysis.
Statistical analysis was performed with
SigmaStat (SPSS, Chicago, Ill.). Comparisons between groups were
performed by two-tailed tests. Comparisons between multiple groups were
performed by analysis of variance (ANOVA), followed by Tukey's test, a
conservative method for the identification of differences between
pairs. Correlations were identified by the Pearson product method.
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RESULTS |
Measuring mycobacterial drug tolerance in BACTEC 12B medium.
The essential features of the use of BACTEC 12B medium to study
mycobacterial killing are illustrated in Fig.
1. In this experiment, M. tuberculosis H37Ra was allowed to grow in BACTEC 12B
medium until the GI reached 250. At that time (day 0), EMB (2.5 µg/ml), INH (0.1 µg/ml), INH-EMB, or RIF (2.0 µg/ml) was added.
These concentrations are routinely used for susceptibility testing in BACTEC 12B medium. GIs were then monitored daily.
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FIG. 1.
Killing of M. tuberculosis H37Ra
by EMB, INH, and RIF in BACTEC 12B medium. INH resistance emerged in
cultures exposed to INH alone. This was prevented by addition of EMB
(upright triangles) without otherwise affecting the rate of killing.
 , no drug;  , EMB;  , INH;
 , INH-EMB;  , RIF.
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GIs in cultures to which drug was not added rose to a maximum of over
600 and gradually declined thereafter. Among cultures
to which drug was
added, the GIs declined the most rapidly in
those with RIF and declined
the least rapidly in those with EMB.
Cultures with INH or INH-EMB
showed intermediate responses that
initially were identical, but those
responses subsequently diverged.
This was not due to the loss of
potency of INH (data not shown)
but, instead, was due to the emergence
of INH resistance, confirmed
by susceptibility testing on agar. This
emergence of resistance
was prevented by the addition of EMB-INH. The
addition of EMB
to INH did not otherwise affect the rate of
sterilization. A two-drug
combination was not required to study killing
by rifampin, as
resistance emerged only in a small number of isolates,
and, even
then, resistance emerged only after 3 weeks of culture. This
may
be due to the low frequency of RIF-resistant colonies in most
susceptible
populations.
14CO
2 production in BACTEC 12B medium reflects
the product of the number of viable organisms and their average state
of metabolic
activity. Declines in GI thus do not a priori indicate
killing.
We therefore experimentally determined the number of viable
bacilli
each day by measuring the time required for subcultures to
become
positive in bottles without drug. As shown in Fig.
2, the number
of days to reach a GI of 30 (days to positivity [DTP]), is inversely
proportional to the
log
10 CFU of the inoculum (log
10 CFU = 5.09
0.257 × DTP;
r2 = 0.99),
indicating that DTP in subcultures can serve as a surrogate
for
conventional CFU determination. The method is more sensitive
than
direct CFU determination and is unaffected by clumping.
Therefore, aliquots of 100 µl were removed daily from cultures to
which drug had been added. Bacilli were sedimented to minimize
drug
carryover; the medium in the original bottle was replenished
with drug
to maintain a constant concentration. The results for
INH and RIF are
indicated in Fig.
3. CFU values were
calculated
from DTP values for subcultures. The correlation between the
GI
and the calculated CFU was statistically significant at
P
equal
to 0.003 for each drug. These experiments indicate that the rate
of killing of
M. tuberculosis in the presence of drugs can
be
determined by monitoring the rate of decline of GI. No such
relationship
was observed for cultures to which no drug was added
(
P = 0.27;
data not shown). This occurred because
subcultures were positive
very quickly (<1 day) due to the large
number of organisms and
also because, late in the stationary phase, the
GI declined somewhat
without a concomitant decline in viability.
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FIG. 3.
Relationship of GI to CFU in cultures with INH (A) or
RIF (B). CFU values were calculated from DTP values for daily
subcultures. The number of viable bacilli in cultures exposed to INH
was reduced by 99% (2 log10) after slightly more than 2 days (A). The corresponding GI at that time was 130.
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Statistical analysis of growth curves generally requires their
reduction to a single number, such as DTP. Killing, like growth,
occurs
as a logarithmic process. In this study, the rate of killing
was
analyzed as the number of days required for 99% sterilization
(days to
sterilization [DTS]). This value rather than the value
for complete
sterilization (less than one viable organisms per
sample) was selected
as it has less inherent variability. Additionally,
there is precedent
for use of a 10
2 proportion in TB drug susceptibility
testing. As indicated in
Fig.
3, 99% sterilization was determined to
occur when the GI
had declined from 250 to 130. The DTS values of
H
37Ra with EMB,
INH, INH-EMB, and RIF were 6.5, 3.3, 3.1, and 1.3 days,
respectively.
Tolerance of clinical isolates.
Tolerance was then examined
for 39 drug sensitive isolates, obtained from a prospective clinical
trial in which two relapses occurred (as reported previously
[21]). Both subjects with relapses had initially
responded, with samples from the subjects becoming culture negative on
agar after days 14 and 30, respectively, and in BACTEC 12B medium after
days 30 and 90, respectively. Cultures on agar again became positive
within several months after treatment had stopped, in association with
clinical evidence of disease. In both cases, cultures in BACTEC 12B
medium were rapidly positive during the last month of therapy; this
reflects the greater sensitivity of BACTEC 12B medium for detection of
small numbers of viable bacilli. Both relapse isolates remained fully
drug sensitive. One subject (subject 29) was documented to have been
noncompliant to the treatment regimen during the last month of therapy,
by pill dispensing and clinic attendance records. The second subject with a relapse appeared to have been fully compliant with the treatment
regimen. A third subject met the definition of treatment failure
(repeated isolation of M. tuberculosis on or after day 120).
This individual was ultimately cured by continued therapy and did not
have a relapse. The rate of all adverse outcomes was 8%.
Pretreatment isolates from these 39 subjects were tested for tolerance
to EMB, INH-EMB, and RIF. All isolates underwent one
additional growth
cycle in fresh BACTEC 12B medium bottles prior
to measurement of
tolerance, to ensure equal logarithmic-phase
growth conditions among
all isolates. As shown in Fig.
4 and
Table
1, the rate of killing by INH and
RIF of isolates from subjects
who relapsed or failed treatment was
markedly delayed compared
to that for other isolates. This indicates
that tolerance may
predict failure and relapse in patients undergoing
treatment for
TB.
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FIG. 4.
Killing of clinical M. tuberculosis isolates,
grouped according to the duration of persistence in sputum during
therapy. Isolates from subjects who had relapses or whose sputum
cultures were persistently positive through day 120 of therapy (black
circles) showed increased tolerance to INH-EMB and RIF compared to all
other isolates.
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To determine whether a more general relationship existed between
tolerance and persistence, subjects were classified according
to the
day of collection of the last sputum specimen to indicate
growth within
20 days after inoculation into BACTEC 12B medium
(last rapidly positive
culture). This threshold was selected on
the basis of the report of
Epstein et al. (
8) indicating a
relationship of this
parameter to the outcome of therapy. The
median day of the last rapidly
positive culture was 30 days (range,
4 to 180 days). Isolates from
subjects with delayed clearance
of viable bacilli from sputum (last
rapidly positive culture on
or after day 90;
n = 7)
were significantly more tolerant than
all others (DTS values, INH-EMB,
5.4 ± 2 versus 4.0 ± 1 [
P = 0.019];
RIF,
2.5 ± 1 versus 1.8 ± 0.3 [
P = 0.017]). In
addition, a linear
relationship between day of last rapidly positive
culture and
days to 99% sterilization in vitro was identified for
INH-EMB
(
P = 0.012) (Fig.
5) and RIF (
P = 0.026)
(data not shown) but
not for EMB (
P = 0.9) (data not
shown). These findings suggest
that the observations regarding the
three failures or relapses
were not likely due to chance.
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FIG. 5.
Correlation between persistence (sputum culture
positivity during therapy) and tolerance to INH (days required for 99%
sterilization).
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There was no apparent relationship between persistence and GI in the
absence of drug, either in the logarithmic phase (before
day 0) or in
the stationary phase (
P = 0.17 to 0.9 (Fig.
4). This
indicates that the growth rate under ideal conditions or that
under
conditions of nutrient depletion does not appear to determine
mycobacterial survival in the presence of drug. This negative
finding
must be taken in context, however, as our preliminary
studies indicate
that GI does not serve as a satisfactory surrogate
for viability during
late-stationary-phase
growth.
As indicated in Table
1, the bactericidal activities of RIF, INH-EMB,
and EMB differed by ANOVA (
P < 0.001). All pairwise
comparisons also showed significant differences (
P < 0.05). Five
isolates were studied with INH alone; these results
did not differ
from those for INH-EMB treatment (
P = 0.22 by paired analysis),
thus confirming the preliminary findings
obtained with H
37Ra.
Mechanisms of tolerance.
To determine whether the rate of
killing was concentration dependent, one clinical isolate (isolate
80730, a tolerant pretreatment isolate from a patient who had a
relapse, despite full compliance) was studied with a 40-fold range of
concentrations of INH (0.025 to 1.0 µg/ml). All concentrations tested
were greater than the MIC of INH for this strain. EMB was added to INH
at a fixed concentration (2.5 µg/ml) to prevent the emergence of INH
resistance. The rate of killing by INH did not vary over this range of
INH concentrations, as indicated in Fig.
6.
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FIG. 6.
Lack of effect of INH concentration on the rate of
killing of a tolerant clinical M. tuberculosis isolate. All
concentrations tested were greater than the MIC for this isolate.
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The majority of INH-sensitive
M. tuberculosis isolates are
highly susceptible (
0.025 µg/ml), but for 40% of isolates MICs
are
0.05 to 0.1 µg/ml (
22). To determine whether differences
in tolerance might be reflected in differences in MIC, the MICs
for
selected isolates were measured in BACTEC 12B medium. The
findings for
INH are shown in Table
2. No relationship
between
tolerance and MIC was identified. This indicates that tolerance
is not likely due to expression of drug-specific factors. Such
a
hypothesis is further supported by the observation that tolerance
to
INH and RIF was highly intercorrelated among the clinical isolates
(Fig.
7) (
P = 0.0002),
despite the marked difference in the mechanisms
of action of these
drugs.
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TABLE 2.
Relationship between MIC of INH, tolerance in vitro, and
persistence in vivo, for four clinical isolates
of M. tuberculosis
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FIG. 7.
Correlation between tolerance to RIF and INH in
drug-sensitive clinical isolates of M. tuberculosis,
measured as DTS.
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In gram-positive bacteria, two forms of tolerance have been described,
and these are based on the characteristics of the fraction
of organisms
that survive drug exposure (
19). In the phenotypic
form of
tolerance, surviving organisms are killed at the same
rate as the
parent strain when re-exposed to drugs. To characterize
the type of
tolerance in
M. tuberculosis, two isolates were exposed
to
INH-EMB in vitro and the surviving bacilli were recovered.
The isolate
from subject 5, a subject with average characteristics,
was exposed for
11 days, and that from subject 80730, a patient
with relapse, was
exposed for 26 days. The surviving bacilli showed
tolerance equal to
that of the parent strain (4.4 versus 4.2 days
for surviving and parent
strains of isolate 5, respectively, and
7.7 versus 8.8 days for
surviving and parent strains of isolate
80730, respectively). The
killing curves for the isolates from
subject 5 are shown in Fig.
8. These data appear to indicate that
tolerance is a stable phenomenon and that within each strain its
expression is phenotypic.
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FIG. 8.
Effect of prolonged drug exposure in vitro on expression
of tolerance. The surviving bacilli of the isolate from subject 5 were
recovered after 11 days of exposure to INH-EMB. The surviving bacilli
exhibited tolerance identical to that of the original isolate.
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The effect of prolonged drug exposure in vivo during therapy was also
studied with isolates from the two subjects with relapses.
The isolates
were obtained after therapy had been initiated. In
the case of subject
80730, the relapse isolate was available for
study. This isolate showed
greater tolerance to INH-EMB than the
original isolate, with the DTS
increasing from 8.8 to 10.9 days.
In the case of subject 29, isolates
were available after 1 week
and 1 month of standard therapy. DTS values
for isolates treated
with INH-EMB increased in a stepwise fashion
during this brief
interval, from 4.2 to 4.5 to 5.1 days. The isolate
also became
progressively more tolerant of EMB, with time to
sterilization
increasing from 8.9 to 15.6 days and, ultimately, to 18.5 days
(Fig.
9). These experiments indicate
that tolerance may arise
in vivo during multidrug therapy.
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FIG. 9.
Effect of prolonged drug exposure in vivo on expression
of tolerance to INH. Isolates were collected from the sputum of subject
29 after 7 and 30 days of standard therapy. Tolerance increased in a
stepwise manner. The subject ultimately had a relapse.
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DISCUSSION |
The eradication of TB has proven to be an elusive goal, often in
individual patients as well as in large populations. It is believed
that most actively replicating bacilli are killed rapidly during the
first few weeks of therapy for TB. Prolonged treatment is required to
eliminate persisting, nonreplicating organisms which exhibit reduced or
otherwise altered metabolic activity and reduced susceptibility to the
bactericidal activities of certain antimycobacterial drugs (11,
24).
This report may shed light on that phenomenon. In it, a method for the
measurement of drug tolerance with the BACTEC TB 460 culture system was
described. Its essential features are as follows: (i) the GI is high at
the time when drug is added, and (ii) the GIs of tolerant isolates
remain high, despite drug exposure. This differs fundamentally from
susceptibility testing, in which the GI is low at the time of drug
addition and in which the GIs of resistant isolates increase, despite
drug exposure. The selection of 250 as the GI threshold at which time
drug is added may have significantly influenced the results. At that
GI, cultures are near the completion of the logarithmic growth phase
and are approaching the stationary growth phase. This may have
emphasized the effects of drug on non-replicating-stage bacilli. This
emphasis may be clinically appropriate, given the significance of
nonreplicating mycobacteria in clinical latency as well as in
persistence during therapy.
Pretreatment isolates from patients who ultimately failed treatment or
who had relapses after standard therapy showed greater tolerance than
those from patients whose treatment was successful. More generally, a
linear relationship between strain tolerance and the duration of
persistence of viable mycobacteria in sputum during treatment was
identified. This is the first study to indicate that a microbial
factor, aside from RIF or multidrug resistance, may affect the outcome
of therapy for TB. Further studies are warranted to verify this
finding, given the small number of adverse outcomes described in this
report. Additional studies are also warranted to determine whether
measurement of strain tolerance can be used prospectively to improve
outcomes and reduce treatment costs. Further studies are also needed to
determine the variability and other potential limitations of the method.
The potential mechanisms for tolerance remain uncertain. The high
correlation in tolerance to RIF and INH is in distinct contrast to
mycobacterial resistance to these drugs. Multidrug resistance is most
often the result of the accumulation of mutations in the genes of drug
targets, sometimes in conjunction with decreased permeability of the
mycobacterial cell wall. The data presented here appear to indicate
that these factors are unlikely to be involved in tolerance. Rather, it
may be more likely that differences between isolates in the time of
transition to the stationary phase of growth may be more relevant.
Further studies will be required to examine this question.
This report also indicates that significant differences exist in the
bactericidal activities of standard anti-TB drugs, with RIF being the
most active, EMB being the least active, and INH being intermediately
active. This ranking is consistent with clinical observations regarding
the sterilizing activities of these drugs (16). In this
context, "sterilizing activity" refers to the ability to clear
viable organisms from sputum rapidly during therapy (17).
RIF and pyrazinamide have high levels of sterilizing activity in vivo.
The incorporation of these drugs into standard anti-TB therapy has
allowed treatment to be shortened from 12 to 6 months and preventive
therapy to be shorted from 9 to 2 months (5, 7, 10, 18, 20).
EMB, in contrast, has little sterilizing activity (6, 24).
Relapse rates increase when EMB is substituted for RIF in the
continuation phase of anti-TB therapy (9). Mitchison (12) has argued that the development of new drugs with high levels of sterilizing activity is critical if anti-TB therapy is to be
shortened and its effectiveness increased. This is the first report to
indicate that a correlate of sterilizing activity can be readily
measured in an in vitro assay. It may indicate an important potential
role for this assay in anti-TB drug development.
The factors that determine the bactericidal activity of a drug are not
known, but one may speculate that its mechanism of action is critical.
EMB acts through the inhibition of synthesis of the arabinan component
of cell wall arabinogalactan (15). This biosynthetic pathway
may not be a critical one in nonreplicating bacilli. In contrast, RIF,
by inhibiting RNA polymerase, may be more likely to affect essential
processes in organisms in all stages of growth, particularly those in
nonreplicating states. This hypothesis is supported by studies that
indicate that drugs that affect cell wall synthesis have markedly
reduced bactericidal activities when they are tested against
non-replicating-stage bacilli, whereas the activities of RIF and
streptomycin are less profoundly affected by the replication stage of
the bacilli (11).
In summary, this report describes a novel method for measurement of the
rate of killing of isolates of M. tuberculosis. The method
may be of significance in the treatment of patients with TB and in the
evaluation of new anti-TB drugs.
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ACKNOWLEDGMENTS |
This study was supported by grants AI45244 and AI41911 from the
National Institutes of Health.
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FOOTNOTES |
*
Corresponding author. Mailing address: Case Western
Reserve University School of Medicine, BRB 1034, 2109 Adelbert Rd.,
Cleveland, Ohio 44106-4984. Phone: (216) 368-4844. Fax: (216) 368-2034. E-mail: rsw2{at}po.cwru.edu.
Present address: Communicable Disease Cluster of the World Health
Organization, Geneva, Switzerland.
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Antimicrobial Agents and Chemotherapy, November 1999, p. 2600-2606, Vol. 43, No. 11
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Copyright © 1999, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
