THE JOURNAL OF INFECTIOUS DISEASES • VOL.

124, SUPPLEMENT • DECEMBER 1971

© 1971 by the University of Chicago. All rights reserved.

Activity of Gentamicin against Mycobacteria In Vitro and against

Mycobacterium tuberculosis in Mice

w. Eugene Sanders, llija Pejovic, Robert Cacciatore,

Henry Valdez, and Frank P. Dunbar
From the State of Florida Department of Health and
Rehabilitative Services, W. T. Edwards Hospital,
Tampa, and Departments of Medicine and
Immunology-Medical Microbiology, University of
Florida College of Medicine, Gainesville, Florida

Gentamicin was shown to be a highly active inhibitor of growth of clinical isolates

of Mycobacterium tuberculosis and other mycobacteria in vitro. Activity was in-
dependent of susceptibility or resistance of the microorganisms to commonly used
antituberculous agents. At a comparable dosage, gentamicin was weakly tuber-

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culostatic and less efficacious than streptomycin in treatment of mice infected
with M. tuberculosis H37Rv. Since gentamicin possesses antituberculous activity,
administration of the drug to patients for other purposes may possibly impede
recovery of mycobacteria from clinical specimens. Further investigation of the
antituberculous activity of gentamicin appears warranted.

Gentamicin is related to other aminoglycosides Drug-susceptibility testing was performed by

(streptomycin, kanamycin, and neomycin) that
possess antituberculous activity. It has been shown
to inhibit a few strains of mycobacteria in vitro
[1, 2]. The present study was designed to compare
the activities of gentamicin, streptomycin and
isoniazid against a variety of mycobacterial strains
in vitro and against Mycobacterium tuberculosis
in mice.
Materials and Methods
M. tuberculosis strains H37Rv and H37RvSm-R,
Mycobacterium scrofulaceum 19981, and Myco-
bacterium fortuitum 6841 were obtained from the
American Type Culture Collection. All other
strains were isolated from patients at the W.T.
Edwards Hospital, Tampa, Florida. All isolates
were maintained on Lowenstein-Jensen medium
at 37 C.
This study was supported in part by grants from the
Schering Corp., Bloomfield, New Jersey. It was also
assisted by an NIH Research Career Development
Award no. l-K03-AI-38636 and a John and Mary R.
Markle Scholarship in Academic Medicine to Dr.
Eugene Sanders.
The authors thank Drs. Lawrence Manni, Nathan
Schneider, and Charles Hartwig for their assistance.
Please address requests for reprints to Dr. Eugene
Sanders, Department of Medicine, University of Florida
College of Medicine, Gainesville, Florida 32601.
the standard technique, previously described for
this laboratory [3]. All strains were subcultured to
Tween-Albumin liquid medium and incubated at
37 C for seven to 10 days. Each culture was mixed
thoroughly (Vortex mixer) once daily. The cul-
tures were then adjusted to comparable optical
density (OD = 0.48 at 475 nm) with Tween-
Albumin liquid to yield approximately 1 mg/rnl
wet weight. One-tenth ml of each culture was then
used as an inoculum for the susceptibility tests.
All in-vitro tests with gentamicin, streptomycin,
and isoniazid were performed using Proskauer
and Beck liquid medium with glycerol. The drugs
were incorporated into the medium by serial two-
fold dilution. Two drug-free control tubes were
included for each strain tested. All cultures were
mixed thoroughly once daily during incubation.
Extent of growth in all cultures was recorded
weekly for four weeks. Results of tests were de-
termined when heavy growth was noted in the
drug-free control cultures (usually at three to
four weeks). The MIC was defined as the lowest
concentration of drug that completely inhibited
visible growth of the microorganisms. Minimal
bactericidal concentration (MBC) was deter-
mined as follows. Aliquots (0.02 ml) from all
clear tubes were subcultured onto Dubos oleic
acid-albumin agar plates and incubated at 37 C
for three weeks. The lowest concentration of drug

833
834 Sanders et al.

00
that completely prevented growth on subculture o '<t ....

was recorded as the MBC. IV

Resistance or susceptibility to the following
agents was determined by noting the presence or
absence of growth, respectively, on medium
7HI0 (Difco) or Lowenstein-Jensen medium con-
taining a single concentration of drug: (l) para-
aminosalicylic acid, 10 ug/rnl; (2) cycloserine, 50
ug/ml; (3) viomycin, 5 ug/ml; (4) ethionamide,
5 ug/ml; (5) ethambutol, 5~g/ml; (6) kana- "'<t
mycin, 5!1g/ml; and (7) capreomycin, 5 ug/ml,
M. tuberculosis H37Rv was chosen for iv in-
fection of CF-l female mice that weighed approx-

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imately 23 g each (Carworth Farms, New City,
N.Y.). Techniques of standardization of inocu-
lum, preparation of tissues, and determination of
colony counts have been described previously [4].
Each of 140 mice was infected with approximately
2.6 X 106 cfu from an eight-day-old Tween-al-
bumin liquid culture. Mice were separated for
treatment into the following groups: isoniazid,
gentamicin, and streptomycin-30 mice each; and
untreated controls-50 mice. All mice were fed
~
Purina Lab Chow and water ad lib. Treatment o
was begun the day after infection and con-
tinued for 90 days. Isoniazid (0.0125%) was
blended with the meal to provide 18-20 mg of
drug/kg body weight per day. Single daily doses
of 25 mg of active drug/kg body weight of genta-
micin and streptomycin were given subcutane-
ously in 0.1 ml distilled water. These doses have
been shown to produce peak levels in serum of
approximately 25 ug/ml within 1 hr in mice
[1, 5]. At 30, 60, and 90 days after infection, 10
mice from each group were sacrificed. Lungs and
spleens were weighed, and macroscopic surface
lesions were enumerated. These organs were
homogenized; aliquots were serially diluted and
plated. Results for each group of mice were ex-
pressed as average number of cfu per whole
organ. Results were comparable when expressed
as cfu/mg of tissue.

Results
Results of in-vitro susceptibility tests with genta-
micin, streptomycin, and isoniazid are shown in
table 1. Gentamicin inhibited growth of each of
the 13 isolates of M. tuberculosis. Activity was
comparable to that of streptomycin against the
streptomycin-susceptible strains. In addition, gen-
Activity of Gentamicin on Mycobacteria
tamicin was active against the three isolates that
were highly resistant to streptomycin. Isoniazid
was a slightly more potent inhibitor of these iso-
lates than the aminoglycosides. M. tuberculosis
H37Rv, used in the animal studies, was inhibited
by 3.12 ug/rnl of streptomycin and gentamicin,
and by 0.4 ug/rnl of isoniazid.
Gentamicin inhibited growth of each of the 20
strains of Mycobacterium kansasii and Mycobac-
terium intracellulare at a concentration of 6.2
ug/rnl or less. It is noteworthy that 17 of these
strains were highly resistant to isoniazid, and
several were resistant to streptomycin. Gentamicin
was highly variable in degree of inhibition of the
Group IV strains. Each of the two aminoglyco-
sides inhibited M. scrofulaceum at a concentration
of Lti ug/ml, whereas isoniazid was inactive.
The MBCs of gentamicin exceeded the MICs
by twofold to eightfold for the six strains tested
(one M. tuberculosis} two M. kansasii, and three
M. intracellulare). The MICs of gentamicin were
examined for mycobacterial strains divided ac-
cording to susceptibility or resistance to each of
the following drugs: para-aminosalicylic acid, cy-
closerine, viomycin, ethionamide, ethambutol,
kanamycin, and capreomycin. The MICs of genta-
micin were similar for strains susceptible or
resistant to each of the seven drugs.
Results obtained following therapy (for 30, 60,
or 90 days) of mice infected with M. tuberculosis
H37Rv, are shown in table 2. Untreated control
animals developed extensive disease, although no
deaths were noted during the 90-day period. Pro-
gression of lung disease and slight regression of
splenic disease were evident between days 30 and
90. Isoniazid completely prevented appearance of
macroscopic surface lesions in the lung and strik-
ingly suppressed the number of organisms recover-
Table 2. Results of therapy of mice infected with Mycobacterium tuberculosis strain H37Rv with gentamicin,
streptomycin, and isoniazid assayed at 30, 60, and 90 days.
Average no. of lung
lesions/animal
Therapy 30 day 60 day 90 day 30 day
None 47 75 81 29.2
Gentamicin 49 65 67 150
Streptomycin 43 46 39 14.7
Isoniazid 0 0 0 1.8
N aTE. Therapy was started the day after intravenous infection. Mice were given 25 rug/kg of gentamicin or strep-
tomycin subcutaneously per day or 18-20 mg /kg of isoniazid daily in the diet. Results are averages for 10 animals
per group. Lung lesions enumerated were macroscopic surface lesions only.
S35
able from both the lungs and spleens. Streptomy-
cin produced progressive suppression of disease in
lungs and spleens during treatment. At 90 days,
decreases of approximately twofold in lesions of
the lung and organisms in the spleens, and 50-fold
in organisms in the lungs, were noted in compari-
son with control animals. Gentamicin was signifi-
cantly less effective in treatment. Disease of the
lungs progressed between days 30 and 90, al-
though less rapidly than in untreated animals. At
90 days, numbers of lesions and viable mycobac-
teria in the lungs were approximately 25 % and
50% less, respectively, than in control animals.
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The drug did not suppress the population of myco-
bacteria in the spleens.
Five isolates, obtained from each group of
animals sacrificed at 90 days, were tested for
susceptibility to gentamicin, streptomycin, and
isoniazid in vitro. Each isolate was as susceptible
to the four drugs as the inoculum used to initiate
infection.
Discussion
Gentamicin inhibited growth in vitro of a variety
of mycobacteria isolated from patients. This activ-
ity appeared to be independent of susceptibility or
resistance of the isolates to isoniazid, streptomycin,
para-aminosalicylic acid, cycloserine, viomycin,
ethionamide, ethambutol, kanamycin, and capreo-
mycin. Each of 13 isolates of M. tuberculosis was
susceptible to 3.1 ug/ml or less of gentamicin.
Since these concentrations were well within levels
achievable in the serum of experimental animals
[1], activity of these drugs was determined in
mice infected with M. tuberculosis H37Rv.
Gentamicin was only weakly tuberculostatic in
mice. In contrast, streptomycin induced a more
Cfu (10 5 ) /whole organ
Lungs Spleens
60 day 90 day 30 day 60 day 90 day
136 353 13.9 4.7 1.3
92.3 198 15.7 2.0 2.1
38.1 7.6 12.0 2.7 0.6
0.005 o 33.0 9.4 0.002
S36
striking regression of disease at equivalent dosage.
The reasons for the difference in efficacy of these
drugs are not readily apparent. The MICs of the
two drugs for the infecting strain were identical.
Equivalent doses were administered, and emer-
gence of resistance to the drug during therapy was
not detected. In previous studies, approximately
equal levels of gentamicin [1] and streptomycin
[5] in the blood have been found following ad-
ministration of equivalent doses to mice. Because
of the chemical similarity of these drugs, organ
distribution is not likely to have differed signifi-
cantly. Degree of binding of gentamicin [6] and
streptomycin [7] by human serum has been shown
to be nearly equal (25% and 33% respectively).
The possibility remains thatserum or other tissues
from infected mice preferentially bind or inacti-
vate gentamicin.
Despite the results of the in-vivo portion of this
study, further investigation of the antituberculous
activity of gentamicin should be conducted. The
degree of in-vitro activity of this drug, especially
against mycobacteria resistant to commonly used
antituberculous agents, is a compelling reason for
further study. Activity should be assayed in other
animal species to exclude the possibility that the
relative lack of activity in mice is species-specific.
In addition, study of the efficacy of this drug at
higher dosage or in combination with other anti-
tuberculous agents would be useful.
Since gentamicin possesses antituberculous ac-
Sanders et al.
tivity, administration of the drug to patients for
other purposes may possibly impede recovery of
mycobacteria from clinical specimens. Physicians
should be alert to this possibility, especially when
use of the drug is contemplated, and active tuber-
culous infection has not been excluded.
References
1. Waitz, J. A., Weinstein, M. J. Recent microbiological
studies with gentamicin. J. Infect. Dis. 1.19:355-
360, 1969.
2. Jedlickova, Z., Sulova, J., Spurna, M. Tuberculo-
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static effect of gentamicin in vitro. J. Hyg. Epi-
demo (Praha) 11:516-517, 1967.
3. Dunbar, F. P., Davis, R., Jeffries, M. B. Drug-
susceptibility testing in tuberculosis. A method
using diluted bacterial suspensions for the indirect
test. Amer. Rev. Tuberc. Pulm. Dis. 77:350-355,
1958.
4. Pejovic, I., Dunbar, F. P., Cacciatore, R. Experi-
mental infections with multiple species of myco-
bacteria. Amer . Rev. Resp. Dis. 98: 111-113,
1968.
5. Zubrod, C. G. Relation of dosage schedule to thera-
peutic efficiency of streptomycin in the treatment
of K. pneumoniae infections in mice. Bull. Johns
Hopkins Hosp. 82:357-365, 1948.
6. Weinstein, M. J. Experimental background and bac-
teriology of gentamicin. In M. Andrial [ed.]
Gentamicin. Schwabe, Basel, 1967, p. 9-18.
7. Weinstein, L. Antibiotics. III. Streptomycin. In L. S.
Goodman and A. Gilman [ed.] The pharmaco-
logical basis of therapeutics. 3rd ed. Macmillan,
New York, 1965, p. 1230-1241.