Microbiol.
Immunol.
Vol. 36 (4), 361-368, 1992
Bacteriostatic and Bactericidal Activity of Antituberculosis Drugs against Mycobacterium tuberculosis, Mycobacterium avium-Mycobacterium intracellulare Complex and Mycobacterium kansasii in Different Growth Phases Sadaaki
YAMORI,*,1,2
Satoshi
and
ICHIYAMA,2
Michio
Kaoru
SHIMOKATA,2
TSUKAMURA1
1Department of RespiratoryDiseases, National Chubu Hospital, Obu, Aichi 474, Japan and
2The First Departmentof Internal Medicine, Nagoya University Schoolof Medicine, Nagoya, Aichi 466, Japan (Accepted for publication, December 19, 1991)
Abstract mycin,
Bacteriostatic enviomycin
avium•\M.
intracellulare
growth
phases.
phases,
except
the
lag
activity
was
complex
than
in
influenced and
bactericidal
log
phase.
M.
the
log
and
by
the
growth
but
some
strains
and
the
activities
of
M. of
isoniazid
complex
and
avium these
the
The of
complex two
of rifampicin,
Mycobacterium
Mycobacterium
tuberculosis
rifampicin.
avium
and
M.
activity
activities
against
activities
isoniazid.
phase
bactericidal
ethambutol
Bacteriostatic
streptomycin The
and
and
drugs
kansasii
were
of the
drugs
were
similar
was
much
less
stationary phase.
M.
and M. were were
by
to
killed
by
isoniazid
was
the
bactericidal
lag
in
isoniazid, strongest.
marked
and the
growth
bactericidal
most
in
different
isoniazid
contrast,
streptomycin
marked
in
different
was
resisted
killed most
in
was of
streptomycin kansasii
In
tuberculosis activity
studied
susceptible
phases.
bactericidal
isoniazid, streptoMycobacterium
tuberculosis,
in
the
activity, enviomycin,
phase.
Bacteriostatic and bactericidal activities of antituberculosis drugs against nontuberculous mycobacteria were reported by several authors (4-6, 14, 15). However, such activity of drugs in different growth phases has not yet been studied. Only one exception is the report of McCarthy (9), who reported that a strain of Mycobacteriumaviumwas susceptible to the bactericidal activity of isoniazid in the lag phase. The purpose of the present study is concerned with the effect of drugs on different growth phases. MATERIALS
AND
METHODS
Strains. The strains used were: Mycobacteriumtuberculosisstrain 05001 (H37Rv) ; Mycobacteriumavium-Mycobacterium intracellularecomplex (M. avium complex), strains 13008 (serotype 20), 13016 (serotype 4), 13034 (serotype 18), 13011 (serotype 16) and 13887 (serotype 14) ; Mycobacteriumkansasii, strains 07002 (ATCC 12478), 07014 and 07018. The M. aviumcomplex and M. kansasii strains were isolated from 361
362
S. YAMORI
ET AL
patients. They were kept until use by freezing at-20 C. The M. aviumcomplex strains were examined for their morphology according to the method of Moehring and Solotorovsky (10). Of the five strains, only strain 13034 formed smooth, flat, transparent colonies and the others smooth, domed, opaque colonies. Media. A modified Dubos liquid medium and Ogawa egg medium were used. To prepare the former, 1.3 g of Difco Bacto Dubos Broth Base (Difco Lab., Detroit, Mich., U.S.A.) were dissolved in 180 ml of distilled water and sterilized by autoclaving at 120 C for 20 min. To this, 20 ml of bovine serum (Eiken Co., Tokyo) were added aseptically. The composition of Ogawa egg medium is as follows: basal solution (1% KH2PO4 and 1% sodium glutamate), 100 ml; whole eggs, 200 ml; glycerol, 6 ml; 2% aqueous solution of malachite green, 6 ml (the pH becomes 6.8 without adjustment). The medium was dispensed in 7 ml aliquots into tubes, 165 by 16.5 mm, and made as slopes by sterilization at 90 C for 60 min. The medium was used for counting the number of colony-forming units (CFU) and for determining minimal inhibitory concentrations (MICs). Antituberculosisdrugs. Antituberculosis drugs were added to media before sterilization. Rifampicin (RFP) (Lepetit, Milan, Italy), isoniazid (INH) (Shionogi Co., Osaka, Japan), streptomycin sulfate (SM) (Meiji Seika, Tokyo), enviomycin sulfate (EVM) (Toyo Jozo, Shizuoka, Japan) and ethambutol (EB) (Kaken, Tokyo) were used. Rifampicin was dissolved in propylene glycol and the others in distilled water. One volume of the drug solutions was added to 100 volumes of Ogawa egg medium before sterilization. Growth curves. Test strains were cultivated on Ogawa egg medium at 37 C for 21 days. Growing colonies were homogenized by shaking with glass beads for 10 min and suspended in a 0.1 % Tween 80 aqueous solution to a concentration of 1 mg wet weight per ml. A 2.0 ml sample of the suspensions was added to 200 ml of Dubos liquid medium in a 500 ml Erlenmeyer flask. The medium was incubated at 37 C in a shaking machine (amplitude 8 cm; 56 strokes per minute). After 0, 1, 3, 5, 7, 10, and 14 days, a 2.0 ml sample of the medium was taken. The sample was diluted by a 0.1% Tween 80 solution to 10-6 by a 0.1% Tween 80 solution. Each 0.02 ml sample of the dilutions was inoculated onto Ogawa egg medium by a spiral loop that can deliver a 0.02 ml sample by one inoculation (11). The tubes inoculated were incubated at 37 C for 21 days and the number of colonies (CFU) was counted. The growth curve was determined by the number of CFU contained in one ml of the medium. Determinationof minimal inhibitory concentration(MIC). Each 2.0 ml sample of the Dubos liquid medium cultures of different growth phases was diluted by 0.1 % Tween 80 solution to 10-7. A 0.02 ml sample of these dilutions was inoculated onto Ogawa egg medium containing a drug or no drug by the spiral loop. The tubes inoculated were stoppered by a gum cap with a 3 min cut in the bottom and incubated at 37 C for 21 days. The MIC was determined as the lowest concentration of drugs in which the growth from 20-100 colonies was completely inhibited (1, 12). This method is principally the same as the proportion method of Canetti et al (1, 2). The drug concentrations used were: rifampicin, streptomycin and enviomycin, 100,
BACTERIOSTATIC
50,
25,
1.6, 0.2, and
12.5,
6.25,
0.8,
0.4,
0.2,
and
0 ƒÊg/ml
0.1
%
In
rifampicin
did
not
growth
death
tubes
were
sample
10,
at
cultures these
number
tericidal
9.0 to
and
incubated
of
The
5,
a
37
of activity
drug-containing
growing was
medium
that
(unpublished
of
sample
24
hr
of
onto
was
and
to
0.1
medium
medium to
1.25,
% and
after the of
and After to
the
for of
in
the
shown
in
Fig.
and
5 ƒÊg/ml.
by
incubation
CFU
following
A
the
0.02
spiral
21
CFU
The
incubation,
10-7.
medium
number
different
the
streptomycin
medium egg
of
give
2.5,
machine.
of
number
bacteria
10 ƒÊ/ml;
0,
Ogawa
ratio
was
susceptibility
egg
solutions
liquid
counted a
the
5,
shaking
Dubos
of Ogawa
liquid
drug 0,
in the
inoculated
as
Dubos
ethambutol,
by
made It
data).
the
isoniazid, and
against
in
was
bacteria. the
exposure
0.4, 0.006,
cultures of
3.2,
0.8,
0.0125,
enhanced
MICs
6.25, 1.6,
0.025,
bacterial
363
12.5, 3.2,
clumping
the
the
25, 6.25,
0.05,
of
agar
ml
colonies
medium
dilution
7H10
1.0
determined
0.1,
the
sample
MYCOBACTERIA
12.5,
0.2,
prevent
ml
diluted
was
25,
to
change
and
C for
0.4,
confirmed not
20 ƒÊ/ml;
were
dilutions
in
ON
ethambutol, 50,
0.8,
mycobacteria
A
rifampicin
0,
bacterial
80 we
of
0 ƒÊg/ml;
the
did
added
concentrations:
enviomycin,
and isoniazid,
order
Tween
activity.
ACTIVITY
1.6,
in
solution
was
0.4,
determinations,
However,
the
phases
0.8, 0 ƒÊg/ml;
solution
aqueous
cause
BACTERICIDAL
tuberculosis,
that
Bactericidal
the
the
80
(7).
80
final
M.
reported
Tween
1.6, and
(for
Tween
previously to
3.2, 0.1,
0 ƒÊg/ml).
by
AND
ml loop.
days.
Bac-
counted
control
in
medium
a (no
drug).
RESULTS
Growth
Curve
Three M.
avium
examples complex
stationary days
growth
and
10-14
of the and
M.
phases days,
growth kansasii
were
curves showed
regarded
are similar as
an
curves.
The
incubation
1. lag,
period
Other the of
strains log
one
of
and day,
the 5-7
respectively.
Minimal InhibitoryConcentrations(MICs) The results of determination of MICs are shown in Table 1. Generally speaking, the MICs were not so much influenced by the growth phase; the MICs determined in different growth phases remained within a range 1: 4. However, the susceptibility of M. tuberculosisto isoniazid changed according to growth phase. The bacteria of the lag phase were much more resistant than those of the other phases. Of five strains of M. aviumcomplex, three strains (13008, 13016, and 13887) were very susceptible to all drugs, whereas the other two (13034 and 13011) were resistant. M. kansasii strains were moderately susceptible, although remained less susceptible than M. tuberculosis. BactericidalActivityagainst the Bacteria in DifferentGrowth Phases The M. activity
results are shown in the Table 2. tuberculosis: Isoniazid and streptomycin on
the
bacteria
of the
lag
and
the
log
exhibited phase.
The
strong activity
bactericidal of
rifampicin
364
S. YAMORI
ET AL
Fig. 1. Growth curves of M. tuberculosis strain 05001 (triangle), M. avium complex strain 13034 (cross), and M. kansasii strain 07002 (circle) determined by the number of colonyforming units per ml of medium.
was inferior to that of isoniazid and streptomycin. Enviomycin showed slight activity and ethambutol did not show such activity. M. avium complex : Only streptomycin and enviomycin were bactericidal. Streptomycin was bactericidal against three strains (13008, 13016, and 13887) and enviomycin against only strain (13887). The activity of streptomycin was the strongest in the lag phase. Enviomycin also acted most strongly in the lag phase. Although strains 13008, 13016 and 13887 were highly susceptible to all drugs in the susceptibility testing, they were resistant to the bactericidal activity of all drugs except streptomycin and enviomycin. Strains 13034 and 13011 were resistant to the bactericidal activity of all drugs. M. kansasii: M. kansasii strains were resistant to the bactericidal activity of all drugs, although they were relatively susceptible in the susceptibility testing. DISCUSSION
We studied the bacteriostatic and bactericidal activities of five drugs in different growth phases of three mycobacteria. The drug concentrations used for studying the bactericidal activity were those attainable in the blood by usual dosage of administration. Previously the growth curve of M. tuberculosiswas reported as linear in Dubos liquid medium when determined by weight per ml (3, 13). However, the curve determined by the number of CFU per ml seemed to be approximate-
BACTERIOSTATIC
AND
BACTERICIDAL
ACTIVITY
ON
MYCOBACTERIA
365
S. YAMORI
366
Table
2.
Bactericidal
ET AL
activities of antituberculosis drugs, ripampicin, isoniazid, bacterium avium-Mycobacterium intracellulare complex and
ly divided into three phases. The results of the study have shown that the bacteriostatic activities of five drugs, except isoniazid on M. tuberculosis,were almost similar even in different growth phases. However, the susceptibility to isoniazid to M. tuberculosisdiffered depending on growth phase. The bacteria of the lag phase were much more resistant to isoniazid than those of the log and the stationary phases. In contrast, the activity of isoniazid against the other two organisms was similar in different growth phases. In the above examinations, a variation of MICs within 1: 4 was regarded as insignificant, because the determination of the MICs by the actual count method (1, 12) may have error of one step (two fold, 1: 2). This error could occur upward or downward. Of the drugs studied, isoniazid, streptomycin and rifampicin were shown to be bactericidal against M. tuberculosisin the concentrations below 10 itgiml. Enviomycin also was slightly bactericidal but ethambutol was not bactericidal in such concentrations. Isoniazid and streptomycin exhibited stronger bactericidal activities against M. tuberculosis than rifampicin. The bactericidal activities of isoniazid and streptomycin were most marked in the log phase. The bacteria of the lag phase were more resistant to such activities of these two drugs than those of the other growth phases. In contrast, M. avium complex and M. kansasii were usually resistant to the bactericidal activity of the drugs. However, some strains of the M. aviumcomplex, which were relatively susceptible to the drugs, were killed by streptomycin and enviomycin. We regarded a reduction of the CFU to 10% or below as an evidence of bactericidal activity. A. avium complex strains of the lag phase seemed to be more susceptible to the bacterial activities of these drugs than those of the log phase. The bacteria of one strain (13034), which were most resistant in susceptibility testings, were killed
BACTERIOSTATIC
streptomycin,
enviomycin,
Mycobacteriumkansasii
with
drug
AND
for
24 hr
in
against
and different
BACTERICIDAL
ethambutol growth
the number
ACTIVITY
against
Myobacterium
ON
MYCOBACTERIA
tuberculosis,
367
Myco-
phasesa)
of CFU
in control
medium.
by rifampicin and isoniazid in their lag phase. In view of the above findings, it is noteworthy that the M. aviumcomplex strains of the lag phase were most susceptible to the bactericidal activity of streptomycin and enviomycin. This finding is quite different from the fact that M. tuberculosiswas more susceptible to the bactericidal activity of isoniazid, streptomycin and rifampicin in the log phase than in the lag phase. Four strains (13008, 13016, 13011, and 13887) of M. avium complex were highly susceptible to rifampicin. Since it was suspected that strains resistant to rifampicin in the susceptibility testings are also resistant to the bactericidal activity of the drug, we used more susceptible strains to rifampicin in this study. However, the bactericidal activity of rifampicin was not observed even in these strains. These findings may be associated with a fact that the eradication of M. avium complex is very difficult in the treatment of infection due to this organism. In this study, it has been shown that streptomycin and enviomycin may be bactericidal against some strains of M. avium complex. This finding suggests that these two drugs may be useful in the treatment of the infection caused by some M. aviumcomplex strains. REFERENCES
1)
Canetti, G., Rist, N., and Grosset, J. 1963. Mesure de la sentibilite du bacille tuberculeux aux drogues antibacillares par la methode des proportions. Rev. Tuberc. Pneumol. 27: 217-272. 2) Canetti, G., Armstrong, A.R., Bartman, K., Cetrangolo, A., Hobby, G.L., Lucchesi, M., Stewart, S.M., Sula, L., Tsukamura, M., and Schmiedel, A. 1966. Recent progress in drug resistance test for tubercle bacilli (major and minor drugs). Bull. Int. Union. Tuberc. 27: 185-225. 3) Fisher, N.W., and Kirchheimer, W.F. 1952. Studies on the growth of mycobacteria. The occur-
368
4)
5)
6)
S. YAMORI
rence of arithmetic linear growth. Am. Rev. Tuberc. 66: 748-761. Heifets, L.B., Iseman, M.D., and Lindholm-Levy, P.J. 1986. Ethambutol MICs and MBCs for Mycobacterium avium complex and Mycobacterium tuberculosis. Antimicrob. Agents Chemother. 30: 927-932. Heifets, L.B., Iseman, M.D., and Lindholm-Levy, P.J. 1988. Combinations of rifampicin or rifabutin plus ethambutol against Mycobacterium avium complex. Bactericidal synergistic, and bacteriostatic additive or synergistic effects. Am. Rev. Respir. Dis. 137: 711-715. Heifets, L.B., and Lindholm-Levy, P.J. 1989. Comparison of bactericidal activities of streptomycin,
7) 8) 9) 10) 11) 12) 13) 14) 15)
ET AL
amikacin,
kanamycin,
and
capreomycin
against
Mycobacterium
avium
and
M.
tuberculosis.
Antimicrob. Agents Chemother. 33 : 1298-1301. Hui, J., Gordon, N., and Kajioka, R. 1977. Permeability barrier to rifampicin in mycobacteria. Antimicrob. Agents Chemother. 21: 773-779. Hurwitz, C., and Silverman, M. 1950. Measurement of growth of tubercle bacilli by means of spectrophotometer. Am. Rev. Tuberc. 62: 87-90. McCarthy, C.M. 1981. Variation in isoniazid susceptibility of Mycobacterium avium during the cell cycle. Am. Rev. Respir. Dis. 123: 450-453. Moehring, J.M., and Solotorovsky, M.R. 1965. Relationship of colonial morphology to virulence for chickens of Mycobacteriumaviumand nonphotochromogens. Am. Rev. Respir. Dis. 92 : 704-713. Tsukamura, M. 1964. Actual count method for the resistance test of tubercle bacilli. Jpn. J. Tuberc. 12 : 46-54. Tsukamura, M. 1988. Evidence suggesting that the life span of Mycobacteriumavium complex is longer than that of other mycobacteria. Microbiol. Immunol. 32: 769-774. Tsukamura, M. 1989. Comparison of the inoculation of mycobacteria by spiral loop and by pipette. Tubercle 70 : 151. Tsukamura, M., and Yamori, S. 1990. Chemotherapeutic regimens for nontuberculous mycobacterial infection based on in vitro susceptibility test results. Kekkaku 65: 349-357. Tsukamura, M., and Yamori, S. 1990. Bactericidal activities of rifampicin, ethambutol, enviomycin and streptomycin on Mycobacterium avium-Mycobacterium intracellulare complex strains. Kekkaku 65: 519-525. (Received for publication, October 11, 1991)
Immunol.
Vol. 36 (4), 361-368, 1992
Bacteriostatic and Bactericidal Activity of Antituberculosis Drugs against Mycobacterium tuberculosis, Mycobacterium avium-Mycobacterium intracellulare Complex and Mycobacterium kansasii in Different Growth Phases Sadaaki
YAMORI,*,1,2
Satoshi
and
ICHIYAMA,2
Michio
Kaoru
SHIMOKATA,2
TSUKAMURA1
1Department of RespiratoryDiseases, National Chubu Hospital, Obu, Aichi 474, Japan and
2The First Departmentof Internal Medicine, Nagoya University Schoolof Medicine, Nagoya, Aichi 466, Japan (Accepted for publication, December 19, 1991)
Abstract mycin,
Bacteriostatic enviomycin
avium•\M.
intracellulare
growth
phases.
phases,
except
the
lag
activity
was
complex
than
in
influenced and
bactericidal
log
phase.
M.
the
log
and
by
the
growth
but
some
strains
and
the
activities
of
M. of
isoniazid
complex
and
avium these
the
The of
complex two
of rifampicin,
Mycobacterium
Mycobacterium
tuberculosis
rifampicin.
avium
and
M.
activity
activities
against
activities
isoniazid.
phase
bactericidal
ethambutol
Bacteriostatic
streptomycin The
and
and
drugs
kansasii
were
of the
drugs
were
similar
was
much
less
stationary phase.
M.
and M. were were
by
to
killed
by
isoniazid
was
the
bactericidal
lag
in
isoniazid, strongest.
marked
and the
growth
bactericidal
most
in
different
isoniazid
contrast,
streptomycin
marked
in
different
was
resisted
killed most
in
was of
streptomycin kansasii
In
tuberculosis activity
studied
susceptible
phases.
bactericidal
isoniazid, streptoMycobacterium
tuberculosis,
in
the
activity, enviomycin,
phase.
Bacteriostatic and bactericidal activities of antituberculosis drugs against nontuberculous mycobacteria were reported by several authors (4-6, 14, 15). However, such activity of drugs in different growth phases has not yet been studied. Only one exception is the report of McCarthy (9), who reported that a strain of Mycobacteriumaviumwas susceptible to the bactericidal activity of isoniazid in the lag phase. The purpose of the present study is concerned with the effect of drugs on different growth phases. MATERIALS
AND
METHODS
Strains. The strains used were: Mycobacteriumtuberculosisstrain 05001 (H37Rv) ; Mycobacteriumavium-Mycobacterium intracellularecomplex (M. avium complex), strains 13008 (serotype 20), 13016 (serotype 4), 13034 (serotype 18), 13011 (serotype 16) and 13887 (serotype 14) ; Mycobacteriumkansasii, strains 07002 (ATCC 12478), 07014 and 07018. The M. aviumcomplex and M. kansasii strains were isolated from 361
362
S. YAMORI
ET AL
patients. They were kept until use by freezing at-20 C. The M. aviumcomplex strains were examined for their morphology according to the method of Moehring and Solotorovsky (10). Of the five strains, only strain 13034 formed smooth, flat, transparent colonies and the others smooth, domed, opaque colonies. Media. A modified Dubos liquid medium and Ogawa egg medium were used. To prepare the former, 1.3 g of Difco Bacto Dubos Broth Base (Difco Lab., Detroit, Mich., U.S.A.) were dissolved in 180 ml of distilled water and sterilized by autoclaving at 120 C for 20 min. To this, 20 ml of bovine serum (Eiken Co., Tokyo) were added aseptically. The composition of Ogawa egg medium is as follows: basal solution (1% KH2PO4 and 1% sodium glutamate), 100 ml; whole eggs, 200 ml; glycerol, 6 ml; 2% aqueous solution of malachite green, 6 ml (the pH becomes 6.8 without adjustment). The medium was dispensed in 7 ml aliquots into tubes, 165 by 16.5 mm, and made as slopes by sterilization at 90 C for 60 min. The medium was used for counting the number of colony-forming units (CFU) and for determining minimal inhibitory concentrations (MICs). Antituberculosisdrugs. Antituberculosis drugs were added to media before sterilization. Rifampicin (RFP) (Lepetit, Milan, Italy), isoniazid (INH) (Shionogi Co., Osaka, Japan), streptomycin sulfate (SM) (Meiji Seika, Tokyo), enviomycin sulfate (EVM) (Toyo Jozo, Shizuoka, Japan) and ethambutol (EB) (Kaken, Tokyo) were used. Rifampicin was dissolved in propylene glycol and the others in distilled water. One volume of the drug solutions was added to 100 volumes of Ogawa egg medium before sterilization. Growth curves. Test strains were cultivated on Ogawa egg medium at 37 C for 21 days. Growing colonies were homogenized by shaking with glass beads for 10 min and suspended in a 0.1 % Tween 80 aqueous solution to a concentration of 1 mg wet weight per ml. A 2.0 ml sample of the suspensions was added to 200 ml of Dubos liquid medium in a 500 ml Erlenmeyer flask. The medium was incubated at 37 C in a shaking machine (amplitude 8 cm; 56 strokes per minute). After 0, 1, 3, 5, 7, 10, and 14 days, a 2.0 ml sample of the medium was taken. The sample was diluted by a 0.1% Tween 80 solution to 10-6 by a 0.1% Tween 80 solution. Each 0.02 ml sample of the dilutions was inoculated onto Ogawa egg medium by a spiral loop that can deliver a 0.02 ml sample by one inoculation (11). The tubes inoculated were incubated at 37 C for 21 days and the number of colonies (CFU) was counted. The growth curve was determined by the number of CFU contained in one ml of the medium. Determinationof minimal inhibitory concentration(MIC). Each 2.0 ml sample of the Dubos liquid medium cultures of different growth phases was diluted by 0.1 % Tween 80 solution to 10-7. A 0.02 ml sample of these dilutions was inoculated onto Ogawa egg medium containing a drug or no drug by the spiral loop. The tubes inoculated were stoppered by a gum cap with a 3 min cut in the bottom and incubated at 37 C for 21 days. The MIC was determined as the lowest concentration of drugs in which the growth from 20-100 colonies was completely inhibited (1, 12). This method is principally the same as the proportion method of Canetti et al (1, 2). The drug concentrations used were: rifampicin, streptomycin and enviomycin, 100,
BACTERIOSTATIC
50,
25,
1.6, 0.2, and
12.5,
6.25,
0.8,
0.4,
0.2,
and
0 ƒÊg/ml
0.1
%
In
rifampicin
did
not
growth
death
tubes
were
sample
10,
at
cultures these
number
tericidal
9.0 to
and
incubated
of
The
5,
a
37
of activity
drug-containing
growing was
medium
that
(unpublished
of
sample
24
hr
of
onto
was
and
to
0.1
medium
medium to
1.25,
% and
after the of
and After to
the
for of
in
the
shown
in
Fig.
and
5 ƒÊg/ml.
by
incubation
CFU
following
A
the
0.02
spiral
21
CFU
The
incubation,
10-7.
medium
number
different
the
streptomycin
medium egg
of
give
2.5,
machine.
of
number
bacteria
10 ƒÊ/ml;
0,
Ogawa
ratio
was
susceptibility
egg
solutions
liquid
counted a
the
5,
shaking
Dubos
of Ogawa
liquid
drug 0,
in the
inoculated
as
Dubos
ethambutol,
by
made It
data).
the
isoniazid, and
against
in
was
bacteria. the
exposure
0.4, 0.006,
cultures of
3.2,
0.8,
0.0125,
enhanced
MICs
6.25, 1.6,
0.025,
bacterial
363
12.5, 3.2,
clumping
the
the
25, 6.25,
0.05,
of
agar
ml
colonies
medium
dilution
7H10
1.0
determined
0.1,
the
sample
MYCOBACTERIA
12.5,
0.2,
prevent
ml
diluted
was
25,
to
change
and
C for
0.4,
confirmed not
20 ƒÊ/ml;
were
dilutions
in
ON
ethambutol, 50,
0.8,
mycobacteria
A
rifampicin
0,
bacterial
80 we
of
0 ƒÊg/ml;
the
did
added
concentrations:
enviomycin,
and isoniazid,
order
Tween
activity.
ACTIVITY
1.6,
in
solution
was
0.4,
determinations,
However,
the
phases
0.8, 0 ƒÊg/ml;
solution
aqueous
cause
BACTERICIDAL
tuberculosis,
that
Bactericidal
the
the
80
(7).
80
final
M.
reported
Tween
1.6, and
(for
Tween
previously to
3.2, 0.1,
0 ƒÊg/ml).
by
AND
ml loop.
days.
Bac-
counted
control
in
medium
a (no
drug).
RESULTS
Growth
Curve
Three M.
avium
examples complex
stationary days
growth
and
10-14
of the and
M.
phases days,
growth kansasii
were
curves showed
regarded
are similar as
an
curves.
The
incubation
1. lag,
period
Other the of
strains log
one
of
and day,
the 5-7
respectively.
Minimal InhibitoryConcentrations(MICs) The results of determination of MICs are shown in Table 1. Generally speaking, the MICs were not so much influenced by the growth phase; the MICs determined in different growth phases remained within a range 1: 4. However, the susceptibility of M. tuberculosisto isoniazid changed according to growth phase. The bacteria of the lag phase were much more resistant than those of the other phases. Of five strains of M. aviumcomplex, three strains (13008, 13016, and 13887) were very susceptible to all drugs, whereas the other two (13034 and 13011) were resistant. M. kansasii strains were moderately susceptible, although remained less susceptible than M. tuberculosis. BactericidalActivityagainst the Bacteria in DifferentGrowth Phases The M. activity
results are shown in the Table 2. tuberculosis: Isoniazid and streptomycin on
the
bacteria
of the
lag
and
the
log
exhibited phase.
The
strong activity
bactericidal of
rifampicin
364
S. YAMORI
ET AL
Fig. 1. Growth curves of M. tuberculosis strain 05001 (triangle), M. avium complex strain 13034 (cross), and M. kansasii strain 07002 (circle) determined by the number of colonyforming units per ml of medium.
was inferior to that of isoniazid and streptomycin. Enviomycin showed slight activity and ethambutol did not show such activity. M. avium complex : Only streptomycin and enviomycin were bactericidal. Streptomycin was bactericidal against three strains (13008, 13016, and 13887) and enviomycin against only strain (13887). The activity of streptomycin was the strongest in the lag phase. Enviomycin also acted most strongly in the lag phase. Although strains 13008, 13016 and 13887 were highly susceptible to all drugs in the susceptibility testing, they were resistant to the bactericidal activity of all drugs except streptomycin and enviomycin. Strains 13034 and 13011 were resistant to the bactericidal activity of all drugs. M. kansasii: M. kansasii strains were resistant to the bactericidal activity of all drugs, although they were relatively susceptible in the susceptibility testing. DISCUSSION
We studied the bacteriostatic and bactericidal activities of five drugs in different growth phases of three mycobacteria. The drug concentrations used for studying the bactericidal activity were those attainable in the blood by usual dosage of administration. Previously the growth curve of M. tuberculosiswas reported as linear in Dubos liquid medium when determined by weight per ml (3, 13). However, the curve determined by the number of CFU per ml seemed to be approximate-
BACTERIOSTATIC
AND
BACTERICIDAL
ACTIVITY
ON
MYCOBACTERIA
365
S. YAMORI
366
Table
2.
Bactericidal
ET AL
activities of antituberculosis drugs, ripampicin, isoniazid, bacterium avium-Mycobacterium intracellulare complex and
ly divided into three phases. The results of the study have shown that the bacteriostatic activities of five drugs, except isoniazid on M. tuberculosis,were almost similar even in different growth phases. However, the susceptibility to isoniazid to M. tuberculosisdiffered depending on growth phase. The bacteria of the lag phase were much more resistant to isoniazid than those of the log and the stationary phases. In contrast, the activity of isoniazid against the other two organisms was similar in different growth phases. In the above examinations, a variation of MICs within 1: 4 was regarded as insignificant, because the determination of the MICs by the actual count method (1, 12) may have error of one step (two fold, 1: 2). This error could occur upward or downward. Of the drugs studied, isoniazid, streptomycin and rifampicin were shown to be bactericidal against M. tuberculosisin the concentrations below 10 itgiml. Enviomycin also was slightly bactericidal but ethambutol was not bactericidal in such concentrations. Isoniazid and streptomycin exhibited stronger bactericidal activities against M. tuberculosis than rifampicin. The bactericidal activities of isoniazid and streptomycin were most marked in the log phase. The bacteria of the lag phase were more resistant to such activities of these two drugs than those of the other growth phases. In contrast, M. avium complex and M. kansasii were usually resistant to the bactericidal activity of the drugs. However, some strains of the M. aviumcomplex, which were relatively susceptible to the drugs, were killed by streptomycin and enviomycin. We regarded a reduction of the CFU to 10% or below as an evidence of bactericidal activity. A. avium complex strains of the lag phase seemed to be more susceptible to the bacterial activities of these drugs than those of the log phase. The bacteria of one strain (13034), which were most resistant in susceptibility testings, were killed
BACTERIOSTATIC
streptomycin,
enviomycin,
Mycobacteriumkansasii
with
drug
AND
for
24 hr
in
against
and different
BACTERICIDAL
ethambutol growth
the number
ACTIVITY
against
Myobacterium
ON
MYCOBACTERIA
tuberculosis,
367
Myco-
phasesa)
of CFU
in control
medium.
by rifampicin and isoniazid in their lag phase. In view of the above findings, it is noteworthy that the M. aviumcomplex strains of the lag phase were most susceptible to the bactericidal activity of streptomycin and enviomycin. This finding is quite different from the fact that M. tuberculosiswas more susceptible to the bactericidal activity of isoniazid, streptomycin and rifampicin in the log phase than in the lag phase. Four strains (13008, 13016, 13011, and 13887) of M. avium complex were highly susceptible to rifampicin. Since it was suspected that strains resistant to rifampicin in the susceptibility testings are also resistant to the bactericidal activity of the drug, we used more susceptible strains to rifampicin in this study. However, the bactericidal activity of rifampicin was not observed even in these strains. These findings may be associated with a fact that the eradication of M. avium complex is very difficult in the treatment of infection due to this organism. In this study, it has been shown that streptomycin and enviomycin may be bactericidal against some strains of M. avium complex. This finding suggests that these two drugs may be useful in the treatment of the infection caused by some M. aviumcomplex strains. REFERENCES
1)
Canetti, G., Rist, N., and Grosset, J. 1963. Mesure de la sentibilite du bacille tuberculeux aux drogues antibacillares par la methode des proportions. Rev. Tuberc. Pneumol. 27: 217-272. 2) Canetti, G., Armstrong, A.R., Bartman, K., Cetrangolo, A., Hobby, G.L., Lucchesi, M., Stewart, S.M., Sula, L., Tsukamura, M., and Schmiedel, A. 1966. Recent progress in drug resistance test for tubercle bacilli (major and minor drugs). Bull. Int. Union. Tuberc. 27: 185-225. 3) Fisher, N.W., and Kirchheimer, W.F. 1952. Studies on the growth of mycobacteria. The occur-
368
4)
5)
6)
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rence of arithmetic linear growth. Am. Rev. Tuberc. 66: 748-761. Heifets, L.B., Iseman, M.D., and Lindholm-Levy, P.J. 1986. Ethambutol MICs and MBCs for Mycobacterium avium complex and Mycobacterium tuberculosis. Antimicrob. Agents Chemother. 30: 927-932. Heifets, L.B., Iseman, M.D., and Lindholm-Levy, P.J. 1988. Combinations of rifampicin or rifabutin plus ethambutol against Mycobacterium avium complex. Bactericidal synergistic, and bacteriostatic additive or synergistic effects. Am. Rev. Respir. Dis. 137: 711-715. Heifets, L.B., and Lindholm-Levy, P.J. 1989. Comparison of bactericidal activities of streptomycin,
7) 8) 9) 10) 11) 12) 13) 14) 15)
ET AL
amikacin,
kanamycin,
and
capreomycin
against
Mycobacterium
avium
and
M.
tuberculosis.
Antimicrob. Agents Chemother. 33 : 1298-1301. Hui, J., Gordon, N., and Kajioka, R. 1977. Permeability barrier to rifampicin in mycobacteria. Antimicrob. Agents Chemother. 21: 773-779. Hurwitz, C., and Silverman, M. 1950. Measurement of growth of tubercle bacilli by means of spectrophotometer. Am. Rev. Tuberc. 62: 87-90. McCarthy, C.M. 1981. Variation in isoniazid susceptibility of Mycobacterium avium during the cell cycle. Am. Rev. Respir. Dis. 123: 450-453. Moehring, J.M., and Solotorovsky, M.R. 1965. Relationship of colonial morphology to virulence for chickens of Mycobacteriumaviumand nonphotochromogens. Am. Rev. Respir. Dis. 92 : 704-713. Tsukamura, M. 1964. Actual count method for the resistance test of tubercle bacilli. Jpn. J. Tuberc. 12 : 46-54. Tsukamura, M. 1988. Evidence suggesting that the life span of Mycobacteriumavium complex is longer than that of other mycobacteria. Microbiol. Immunol. 32: 769-774. Tsukamura, M. 1989. Comparison of the inoculation of mycobacteria by spiral loop and by pipette. Tubercle 70 : 151. Tsukamura, M., and Yamori, S. 1990. Chemotherapeutic regimens for nontuberculous mycobacterial infection based on in vitro susceptibility test results. Kekkaku 65: 349-357. Tsukamura, M., and Yamori, S. 1990. Bactericidal activities of rifampicin, ethambutol, enviomycin and streptomycin on Mycobacterium avium-Mycobacterium intracellulare complex strains. Kekkaku 65: 519-525. (Received for publication, October 11, 1991)
