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Bacteriostatic and bactericidal activity of ciprofloxacin and ofloxacin against mycobacterium tuberculosis and mycobacterium avium complex
Tubercle (1987) 68, 0 Longman Group
267-276 UK Ltd. 1987
BACTERIOSTATIC AND BACTERICIDAL ACTIVITY OF ClPROFLOXAClN OFLOXACIN AGAWST MYCOSAC7’ERWW TURERCULOSIS AND MYCOBAC77iRlUMAVlUMCOIIIIPLEX*
AND
Leonid B. Heifetst Department
of Medicine,
National
Jewish
Center for Immunology
and Respiratory
Medicine,
Denver,
Colorado
Pamela J. Lindholm-Levy Department
of Microbiology
and Immunology,
School of Medicine, Denver, Colorado
University
of Colorado,
Health Sciences
Center,
Summary MlCs of ciprofloxacin were lower than MlCs of ofloxacin for both M. tuberculosis and M. avium complex strains. The MlCs of both drugs for 41 M. tuberculosis strains were in a narrow range, and significantly lower than the achievable serum concentrations. The MlCs of ciprofloxacin for 46 M. avium strains were in a wide range, and in only 28 % were the broth-determined MlCs of ciprofloxacin within the and lower than the achievable serum level. When same range as for M. tuberculosis compared with the concentrations achievable in macrophages, the brothdetermined MlCs of ciprofloxacin were lower than this level for 61 % of M. avium strains. Both drugs were bactericidal against M. tuberculosis and M. avium, with a low MIUMBC ratio.
R6sumf5 Les CIM de ciprofloxacine ktaient plus basses que celles d’ofloxacine aussi bien pour M. tuberculosis que pour les souches due complexe M. avium. Les CIM des deux mkdicaments pour 41 souches de M. tuberculosis ktaient tr&s prochers les unes des autres et significativement plus basses que les concentrations que I’on peut obtenir dans le s&urn. Les CIM de ciprofloxacine pour 46 souches de M. avium montraient une dispersion large et pour 28 % seulement les CIM de ciprofloxacine d&erminkes en bouillon de culture se situaient dans les m&mes limites de dispersion que celles pour M. tuberculosis et elles dtaient en-dessous du niveau que I’on peut obtenir dans le s&urn. Pour 61 % des souches de M. avium, les CIM de ciprofloxacine mesurks en bouillon de culture dtaient plus bas que les concentrations que I’on peut obtenir dans les macrophages. Les deux produits 6taient bactrkicides contre M. tuberculosis et M. avium, avec un ratio CIMKBM bas.
Resumen Las MlCs de ciprofloxacina eran inferiores a las de ofloxacina tanto para M. tuberculosis coma para cepas del complejo M. avium. Las MlCs de ambos medicamentos para 41 cepas de M. tuberculosis eran muy prdximas entre ellas y significativamente m8s bajas que las concentraciones obtenibles en el suero. Las *Supported by contract NOl-Al-42544 with the National Institute of Allergy and Infectious Disease. Wequests for reprints should be addressed to Leonid 6. Heifets, M.D., National Jewish Center for Immunology Respiratory Medicine, 1400 Jackson St., Denver, Colorado 80206, U.S.A.
and
268
Heifets
and
Lindholm-Levy
MlCs de ciprofloxacina para 46 cepas de M. avium mostraban una amplia dispersion y solo en el 28 % las MlCs de ciprofloxacina determinadas en caldo de cultivo se situaban dentro del mismo rango de dispersion que las observadas para M. tuberculosis y por debajo del nivel obtenible en el suero. En el 61 % de las cepas de M. avium, las MlCs de ciprofloxacina determinadas en caldo de cultivo eran inferiores a las concentraciones obtenibles en macrofagos. Ambos productos eran bactericidas contra M. tuberculosis y M. avium, con bajo coeficiente MIUMBC.
Introduction The renewed interest in the 4-quinolones class of antimicrobial agents during the last few years has led to the development of an increasing number of new drugs with very low minimal inhibitory concentrations (MIC) against a variety of bacterial species. The feasibility of achieving adequate serum concentrations with these new drugs made the use of these quinolones possible for treatment of infections other than urinary tract infections. This is an important difference between the modern 4-quinolones and their predecessors, nalidixic acid and other heterocyclic carbonic acid derivatives. Some of these 4-quinolones have already been tested against some mycobacterial species [l-71. Special attention has been given to two drugs with a very wide spectrum: ciprofloxacin [81 and ofloxacin [9]. It has been shown [I] that MlCs of ciprofloxacin were significantly lower than MlCs of norfloxacin in agar dilution tests with M. tuberculosis, M. avium, M. kansasii, M. chelonae, and M. fortuitum. Ofloxacin (DL8280) produced an inhibitory and bactericidal effect against M. tuberculosis in vitro 121 and was found effective in single-drug chemotherapy of 19 patients who were ‘treatment-failure’ cases and had chronic cavitating tuberculosis with multiple drug resistance to the standard antituberculosis drugs [31. The aims of the present study were to determine the MICs, MBCs and MIUMBC ratios of ofloxacin and ciprofloxacin against M. tuberculosis and M. avium complex.
Materials Antimicrobial
and methods
agents
Ciprofloxacin was obtained from Miles Pharmaceuticals, Ofloxacin (Daiichi Pharmaceutical Co.) was obtained from Dr M. Tsukamura. A 1280 yg/ml solution of each drug was made in distilled water for irrigation (Abbott Laboratories) and sterilised by filtration through a Nalgene filter unit (Nalge Company). Serial doubling dilutions of the stock solution were made in sterile distilled water for irrigation, to provide a range of concentrations from 1280 pg/ml to 5 pg/ml. Aliquots of each concentration were stored at -70 “C. Cultures Forty-six strains of the M. avium complex (i.e. M. avium and M. intracellulare) isolated from patients with disseminated infections were used in this study. Smooth transparent colonies from 7Hll agar plates were subcultivated in 7H9 broth. From this point on, references in this paper to M. avium assume M. avium complex. Forty clinical isolates and H& strain of M. tuberculosis were tested. Twenty-three strains had previously been found to be resistant, by the conventional proportion susceptibility test, to at least one antituberculosis drug. Seventeen strains were susceptible to all the antituberculosis drugs. These strains were subcultivated in 7H9 broth. Aliquots of the M. avium and M. tuberculosis broth cultures were kept at -70°C.
Ciprofloxacin M/C
determination
in 7H1.2 broth
by the radiometric
and ofloxacin
269
method
7H12 broth, which contains “C-labelled palmitic acid as a source of carbon, was used. Growth leads to degradation of the palmitic acid and release of 14C02 into the atmosphere above the medium. The BACTEC TB-460 instrument (Johnston Laboratories) detects the amount of 14C02 and records it on a scale of zero to 999 as the Growth Index (GI). Vials were tested daily on the BACTEC instrument and the GI was recorded. Before testing M. avium, an initial vial of 7H12 medium was inoculated with a I:50 dilution of a fresh 7H9 broth culture adjusted to the optical density of a No. 1 McFarland standard. When the GI of the vial reached 999, the broth was diluted 1 :lOO, and 0.1 ml was used to inoculate each of a set of test vials. This inoculum provided a bacterial concentration of IO4 to IO5 CFU-ml (11). lnoculum of M. tuberculosis was prepared by seeding a 7H12 vial with a I:2 dilution of a fresh 7H9 broth culture adjusted to equal a No. 1 McFarland standard. When growth in this vial reached GI 400 to 500,O.l ml of the undiluted broth was inoculated into each of a set of test vials, providing IO4 to IO5 CFU/ml. Two drug-free controls were used with each set of tests for both species. One control was inoculated in the same way as the test vials, the second was inoculated with a 1 :I00 dilution of the inoculum to represent 1 % of the bacterial population. M. avium strains were first tested against 16 pg/ml of each drug. Strains susceptible to this concentration were tested against 8.0, 4.0, 2.0, and 1.0 pg/ml to determine the actual MIC. The narrow range of MlCs against M. tuberculosis made it possible to test 2.0, 1 .O, and 0.5 pg/ml of each drug in a primary titration. The concentration of drug producing a daily GI increase and final GI reading lower than those in the 1 :I00 control was considered to have inhibited more than 99 % of the bacterial population and was therefore defined as the MIC. The tests were carried out for no less than 4 or more than 8 days with M. avium, or no more than 10 days with M. tuberculosis. The final day of observation with M. avium was that on which the GI of the undiluted control reached 999. For M. tuberculosis the endpoint of the experiment came when the GI of the 1 :I00 control was greater than 30 for 2 consecutive days. M/C determination
in 7H12
broth
by plating
The determination of MIC by plating was carried out by the same technique as previously described except that duplicate vials were used. Samples were taken from alternate vials on various days and diluted appropriately, based on the GI reading, so as to have 50-500 CFU per plate. The dilutions were inoculated onto 7Hll ‘agar plates, 0.5 ml per plate, and distributed by tilting the plate. Incubation was at 37 “C in 5 % CO2 for 12-14 days, after which the colonies were counted, The MIC was the lowest concentration that inhibited more than 99 % of the bacterial population in broth for up to 8 days.
M/C determination
by the agar dilution
method
Quadrant petri dishes were prepared with 7Hll agar so as to have a different concentration of drug in each of three quadrants; the fourth quadrant was the drug-free control. Against M. avium strains, concentrations of 0.5-l 6.0 pg/ml of each drug were used while 0.5, 1 .O and 2.0 pg/ml of each drug were tested with M. tuberculosis. Each quadrant was inoculated with 0.1 ml of a bacterial suspension. For M. avium the inoculum was either a lop4 dilution of a 7H12 broth culture that had reached GI 999, or a 1O-5 dilution of a 7H9 broth culture adjusted to the optical density of a No. 1 McFarland standard. For M. tuberculosis the
270
Heifets
and
Lindholm-Levy
inocuium was a IO-* dilution of a 7H12 broth culture that had reached GI 400 to 500. These inocula contained 200-300 CFU. The plates were incubated at 37 “C in the presence of 5 % COz for 12-14 days. The lowest concentration of drug that inhibited more than 99 % of the bacterial population on agar plates was considered to be the MIC. MBC
determination
7H12 vials were inoculated with the organism in the same way as described above for radiometric MIC determination, except that duplicate vials were used and a 1 :I00 control was eliminated. Beginning with an inoculum of IO4 to IO5 CFU/ml, the cultures were allowed to incubate until growth was in the exponential phase and the number of CFU/ml was IO5 to 106. Previous studies [IO-123 showed that for M. avium this bacterial concentration occurred when the GI was approximately 400. At this when the GI was 20-80, and for M. tuberculosis time the drugs were added to achieve 1, 2, 4 and 8 times the previously determined MIC. Samples were taken from alternate vials at various time points, diluted based on the GI reading, and 0.5 ml of the dilutions was inoculated onto 7Hll agar plates as previously described. Incubation was at 37 “C for 12-14 days, after which the colonies were counted and CFU/ml calculated. The MBC was the lowest concentration of drug that killed more than 99.9 % of the bacterial population present when the drugs were added. MIUMBC
ratios
Six strains of M. avium and three strains of M. tuberculosis were tested simultaneously for MIC and MBC of both drugs by the plating methods previously described. The ratio of MIC to MBC was then calculated.
Results Growth Curves in 7H12 broth We confirmed in this study our previous finding [IO, 11, 121 that the maximum available numbers of CFU/ml in 7H12 broth were: for M. tuberculosis IO6 to IO7 CFU/ml, and for M. avium, IO* to IO’ CFU/ml. It is known that the time required to achieve maximum growth in 7H12 broth depends on the inoculum size [13]. In this study, the initial concentration of bacteria in 7H12 broth by the procedure described was between IO4 and IO5 CFU/ml. Under these conditions the maximum growth for most M. tuberculosis strains was achieved within 7-10 days, with the exception of some strains with multiple drug resistance, which produced poor growth in this and any other media. In experiments with M. avium the maximum growth was achieved within 4-8 days. The average generation time during the exponential phase of growth was: for M. tuberculosis strains, 38-48 hours; for M. avium strains, 9-12 hours. Examples of growth curves of M. tuberculosis and M. avium are presented in Figure 1 (curves Al). The exponential phase of growth in drug-free medium (curves Al) was detected in this experiment for M. tuberculosis from the third to the eighth day, and for M. avium from the second to the fifth day of incubation. The daily GI reading curves (Bl in Fig. 1) correlated well with the growth curves (Al) and M. avium. These curves also during the exponential phases for both M. tuberculosis correlated well in the presence of ciprofloxacin (A3 & 83) and ofloxacin (A4 81 B4). These experiments, conducted with three M. tuberculosis and six M. avium strains, confirmed that the inhibition of GI in vials containing ciprofloxacin or ofloxacin reflected actual inhibition of growth by these drugs. These data correspond with previous findings in determining the MlCs for rifabutine. (ansamycin LM427) [I 1 I and ethambutol [I 21. This justified the use of the radiometric method for determination of MlCs of ciprofloxacin and ofloxacin in liquid medium for the remaining 38 strains of M. tuberculosis and 40 strains of M. avium.
Ciprofloxacin
and ofloxacin
271
8 J& tuberculosis
7
A
E
tuberculosis
1 lvllaviYm/_.
2
Days
4
,
6
6
Days
Figure 1. Examples of correlation between the growth curves (CFU per ml) (A) and the daily GI reading curves (6) in experiments for determination of the MlCs of ciprofloxacin and ofloxacin. l-undiluted control; Z-1:100 control; 3-in presence of ciprofloxacin (MIC); 4-in presence of ofloxacin (MIC).
Minimal
inhibitory
concentrations.
MlCs of ciprofloxacin for M. tuberculosis strains were between 0.25 and 2.0 yglml when determined in 7H12 broth radiometrically and 0.125 to 2.0 pg/ml when determined in 7Hll agar plates (Table I). The MlCs of ciprofloxacin for M. avium were significantly higher when determined either by the broth (radiometric) or agar dilution method. Another difference was that the range of MlCs for M. avium was much wider than for M. tuberculosis. For only some of M. avium strains was the MIC as low as the MIC range for M. tuberculosis. No significant differences were found between broth- and agar-determined ciprofloxacin MlCs for M. tuberculosis, but the broth-determined MlCs were lower than agar-determined MlCs for most M. avium strains (Fig. 2, A). The MlCs of ofloxacin for M. tuberculosis were in a narrow range of 0.25 to 2.0 pg/ml when determined in 7H12 broth (Table II), which is the same as for ciprofloxacin. The agardetermined MlCs of ofloxacin were between 0.5 and 1.O yglml. The MlCs of ofloxacin for M. avium strains were much higher than for M. tuberculosis (Table II), and ranged between 4.0 and more than 32.0 pg/ml by either method. The broth- and agar-determined MlCs of ofloxacin had the same value for most of the M. tuberculosis strains; the broth-determined MlCs were slightly lower than agar-determined for about half of the M. avium strains (Fig. 2, B).
272
Heifets
Table I.
and Lindholm-Levy
MlCs of ciprofloxacin Organism
for 41 M. tuberculosis M/C
M. fuberculosis M. avium
fjcg/mlI
in 7Hll
agar
Range
50 %
90
0.125-2.0 1.1X32.0
0.35 8.0
0.48 23.0
M. Aviun -7Hl2 32.0
2
8.0
.
i/
I c
4.0 2.0
-
1.0
m
e
-
.
%
(pg/mll
in 7H72
broth
Range
50
90
0.25-2.0 0.5-16.0
0.34 3.0
0.78 22.0
.
I
%
%
CC@BJX
2.0
4.0
i
/ .
-
.
.
7Hll
AGAR 6.0
16.0
.
.
7Hll
.
1.0
M/C
strains.
A
BROTH
16.0
-i
and 46 M. avium
:/
AGAR
32.0
1.0
2.0
4.0
6.0
16.0
32.0
uglml
lrdml M
tuberculosis 7H12 BROTH
B /
2.0l.O-e s
7Hll AGAR 0.125
0.25
0.5
1.0
2.0
Figure 2. MlCs of ciprofloxacin (A) and ofloxacin 40 M. tuberculosis and 46 M. avium strains.
Minimal
bactericidal
concentrations
t/,1,
4.0
(6) determined
and MWMBC
0.125
0.25
,:“p:” 0.5
1.0
2.0
by 7H12 broth and 7Hll
4.0
agar plate methods
for
ratios
In experiments conducted in 7H12 broth, the MBCs were determined on the basis of comparisons between the kinetics of the number of CFU/ml in drug-free medium and in the presence of different concentrations of drugs added on the 2nd to the 8th day of cultivation when the radiometric readings indicated that the culture was in an exponential phase and the number of CFU/ml was above 105. The actual determination of the number of CFU/ml in broth by plating culture samples, confirmed that the number of CFU/ml when the drug was
Ciprofloxacin Table II.
MlCs of ofloxacin Organism
MIC
M. tuberculosis M. avium
Table
III.
for 41 M. tuberculosis
Bactericidal
in 7till
agar
M/C
Range
50
90
Range
50
0.5-l .o 4.0->32.0
0.5 32.0
0.26-2.0 4.0->32.0
0.7 12.0
activity
Organism
(hg/mll
Table
%
1 .o >32.0
effect
CFlJ/ml when drug added
H3,Rv
%
MBC
By CFlJ/ml
Wml)
6.4~10~ 1.5x105 8.9x IO5
2.0 2.0 2.0
1.0 1 .o 0.5
M. M. M. M. M. M.
3.6~10~ 2.0x 105 4.8x lo5 3.4x lo5 2.0x lo5 1.4x105
4.0 8.0 8.0 16.0 32.0 64.0
4.0 1.0 1 .o 8.0 16.0 16.0
IV.
3350-4 1017-5 4534 3337-4 3349-4 168-4
Bactericidal
activity
Organism
avium avium avium avium avium
M. avium
%
90
%
1.4 28.0
Ratio MIUMBC
Radiometrically 1.0 1.0 0.5 4.0 1.0 1.0 8.0 16.0 16.0
6.4x lo5 l.5X105 8.9x 10s
M/C fug/ml) in the same experiment
effect
CFWml when drug added
M. M. M. M. M.
in 7H1.2 broth
1:2 I:2 1:4 1:1 1:8 1~8 1:2 1:2 I:4
of ofloxacin.
Bactericidal
H3,Rv M. tuberculosis M. tuberculosis#3105 M. tuberculosis#2923
strains.
MIC lpg/ml) in the same experiment
M tuberculosis#3105 M. tuberculosis#2923 avium avium avium avium avium avium
/ug/ml)
273
of ciprofloxacin.
Bactericidal
M. tuberculosis
and 46 M. avium
and ofloxacin
MBC
By CFlJ/ml
Wmll 2.0 2.0 2.0
Ratio MIUMBC
Radiometrically
1 .o 1.0 0.5
1.0 1.0 0.5
I:2 1:2 114
1017-5 453-4 3350-4 3337-4 3349-4
2.0x105 4.8~10~ 3.6x lo5 3.4x 10s 2.0x105
16.0 16.0 64.0 64.0 256.0
4.0 4.0 16.0 16.0 64.0
4.0 4.0 16.0 16.0 64.0
1:4 114 1:4 1:4 1:4
168-4
1.4x105
512.0
32.0
32.0
1:16
added was: for M. tuberculosis, from 1.5x lo5 to 8.9x 1Or’; and for M. avium, from 1.4x IO5 to 4.8~10’ (Tables III and IV). To have the most accurate MIUMBC ratios, the MlCs were determined by two methods with the same strains: by sampling and plating and by radiometric GI readings as previously described. Unlike the MBC determination, the drugs were added at the beginning of cultivation, when the concentration of bacteria was between
274
Heifets
and
Lindholm-Levy
IO4 and IO5 CFU/ml. In experiments with three strains of M. tuberculosis and six strains of IM. avium we found no difference in the MIC values determined either by sampling (CFU/ml] or GI radiometric readings (Tables III and IV]. The MBC of ciprofloxacin (Table Ill] was 2.0 pg/ml for all three strains of N1. tuberculosis, and between 4.0 and 64.0 ug/ml for six M. avium strains. The low MIC/MBC ratios indicated high bactericidal activity: 1:2-l :4 for M. tuberculosis, and 1:2-I:8 for M. avium. The MBCs of ofloxacin (Table IV) were the same as those of ciprofloxacin (2.0 mg/ml) for M. tuberculosis strains, but were substantially higher for M. avium strains (from 16.0 to 512.0 kg/ml). The MIC/MBC ratios were I:2 to I:4 for M. tuberculosis, and I:4 for five of six strains of M. avium. Discussion The mean serum peak level (C,,,) of ciprofloxacin is between 2.0 and 3.0 pg/ml attainable 1.25 h after an oral administration of 500 mg [14-171 with good penetration into different tissues [14, 16, 171. The concentration in sputum from patients with cystic fibrosis is 1 .Ol f0.24 pg/ml [181. The peak levels in serum are higher after a 750 mg oral dose [19]. The pharmacokinetics of ofloxacin have similar features, with a peak serum level of 10.7 pg/ml after administration of 600 mg orally [20]. The MlCs of both ciprofloxacin and ofloxacin for M. tuberculosis were much lower than the achievable serum and tissue levels per site. The MlCs of ciprofloxacin for 90 % of M. tuberculosis strains (MIC& were 0.48 pg/ml or less when determined in 7H12 broth. These data correspond with previous findings: 1 pg/ml [I] and 0.5 pg/ml [5] when the MIC was determined in 7HlO agar plates. The MI&, of ofloxacin against M. tuberculosis in this study was less than 1 .O yg/ml when determined in 7Hll agar plates, and 1.4 yglml in 7H12 broth, with a range of 0.5-1.0 and 0.25-2.0 ug/ml, respectively. These data also correspond to findings by other authors: 0.67-1.25 yglml in Ogawa medium [2], and 0.5-1.0 pg/ml in 7HlO agar plates 151. Comparisons of in vitro activities of the two quinolones in this, as well as in a previous study [51, indicate that the MlCs of ciprofloxacin were lower than MlCs of ofloxacin (see also Fig. 2). The MlCs of ciprofloxacin and ofloxacin for M. avium complex strains were substantially higher than for M. tuberculosis, and were in a wide range, regardless of the method of determination (in 7Hll agar plates or in 7H12 broth). For only 13 of 46 M. avium strains were the broth determined MlCs of ciprofloxacin within the same range as for M. tuberculosis (2.0 pg/ml or less). There were no strains of M. avium for which the MlCs of ofloxacin were within this range. Ciprofloxacin is probably more active than ofloxacin against M. avium. In general, both drugs are less promising against M. avium than against M. tuberculosis, because the MlCs against the former were, for many strains, higher than the achievable serum concentrations. At the same time, the following two facts should be taken into account: concentrations of ciprofloxacin in macrophages are two to three times higher than in the extracellular fluid [21], and ciprofloxacin has a wide range of MlCs against M. avium. The broth-determined MlCs of ciprofloxacin for more than half of M. avium strains (28 of 46) were 4.0 pg/ml or less, lower than the concentrations achievable within the macrophages. This fact indicates that ciprofloxacin may be effective in treatment of those patients whose M. avium isolate is susceptible to low concentrations of the drug and that an MIC should be determined against each strain, preferably in broth, which is a more sensitive technique, providing more accurate information. The perspective of practical application of ciprofloxacin may be broadened should the synergistic or additive effect shown with other drugs apply as well to ciprofloxacin, lowering its MIC against M. avium strains at least twofold.
Ciprofloxacin
and ofloxacin
275
Another important feature of ofloxacin and ciprofloxacin, confirmed in this study, is the high bactercidal activity of these drugs, with MIUMBC ratios of I:2 to 1:4 for both drugs against M. tuberculosis, 1:1-l :8 for ciprofloxacin, and I:4 for ofloxacin against M. avium.
Acknowledgements This work was supported by contract No. l-Al042544 with the National Institute of Allergy and Infectious Disease. We thank Dr M. Tsukarmura for providing us with ofloxacin for this work, Miles Laboratories for supplying us with ciprofloxacin, B. Silverstein for the artwork, and C. J. Queen for preparation of the manuscript.
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and
Lindholm-Levy
19 Gonzalez, M. A., Uribe, F., Moisen, S. 0.. Fuster, A. P., Selen, A., Welling, P. G., Painter, 6. (1984). Multiple-dose pharmacokinetics and safety of ciprofloxacin in normal volunteers. Antimicrobial Agents and Chemotherapy, 26, 741. 20 Lockley M. FL, Wise, R., Dent, J. (1984). The pharmacokinetics and tissue penetration of ofloxacin. Journal of Antimicrobial Chemotherapy, 14, 647. 21 Easmon, C. S. F., Crane, J. P. (1985). Uptake of ciprofloxacin by macrophages. Journal of Clinical Pathology, 38, 442.
267-276 UK Ltd. 1987
BACTERIOSTATIC AND BACTERICIDAL ACTIVITY OF ClPROFLOXAClN OFLOXACIN AGAWST MYCOSAC7’ERWW TURERCULOSIS AND MYCOBAC77iRlUMAVlUMCOIIIIPLEX*
AND
Leonid B. Heifetst Department
of Medicine,
National
Jewish
Center for Immunology
and Respiratory
Medicine,
Denver,
Colorado
Pamela J. Lindholm-Levy Department
of Microbiology
and Immunology,
School of Medicine, Denver, Colorado
University
of Colorado,
Health Sciences
Center,
Summary MlCs of ciprofloxacin were lower than MlCs of ofloxacin for both M. tuberculosis and M. avium complex strains. The MlCs of both drugs for 41 M. tuberculosis strains were in a narrow range, and significantly lower than the achievable serum concentrations. The MlCs of ciprofloxacin for 46 M. avium strains were in a wide range, and in only 28 % were the broth-determined MlCs of ciprofloxacin within the and lower than the achievable serum level. When same range as for M. tuberculosis compared with the concentrations achievable in macrophages, the brothdetermined MlCs of ciprofloxacin were lower than this level for 61 % of M. avium strains. Both drugs were bactericidal against M. tuberculosis and M. avium, with a low MIUMBC ratio.
R6sumf5 Les CIM de ciprofloxacine ktaient plus basses que celles d’ofloxacine aussi bien pour M. tuberculosis que pour les souches due complexe M. avium. Les CIM des deux mkdicaments pour 41 souches de M. tuberculosis ktaient tr&s prochers les unes des autres et significativement plus basses que les concentrations que I’on peut obtenir dans le s&urn. Les CIM de ciprofloxacine pour 46 souches de M. avium montraient une dispersion large et pour 28 % seulement les CIM de ciprofloxacine d&erminkes en bouillon de culture se situaient dans les m&mes limites de dispersion que celles pour M. tuberculosis et elles dtaient en-dessous du niveau que I’on peut obtenir dans le s&urn. Pour 61 % des souches de M. avium, les CIM de ciprofloxacine mesurks en bouillon de culture dtaient plus bas que les concentrations que I’on peut obtenir dans les macrophages. Les deux produits 6taient bactrkicides contre M. tuberculosis et M. avium, avec un ratio CIMKBM bas.
Resumen Las MlCs de ciprofloxacina eran inferiores a las de ofloxacina tanto para M. tuberculosis coma para cepas del complejo M. avium. Las MlCs de ambos medicamentos para 41 cepas de M. tuberculosis eran muy prdximas entre ellas y significativamente m8s bajas que las concentraciones obtenibles en el suero. Las *Supported by contract NOl-Al-42544 with the National Institute of Allergy and Infectious Disease. Wequests for reprints should be addressed to Leonid 6. Heifets, M.D., National Jewish Center for Immunology Respiratory Medicine, 1400 Jackson St., Denver, Colorado 80206, U.S.A.
and
268
Heifets
and
Lindholm-Levy
MlCs de ciprofloxacina para 46 cepas de M. avium mostraban una amplia dispersion y solo en el 28 % las MlCs de ciprofloxacina determinadas en caldo de cultivo se situaban dentro del mismo rango de dispersion que las observadas para M. tuberculosis y por debajo del nivel obtenible en el suero. En el 61 % de las cepas de M. avium, las MlCs de ciprofloxacina determinadas en caldo de cultivo eran inferiores a las concentraciones obtenibles en macrofagos. Ambos productos eran bactericidas contra M. tuberculosis y M. avium, con bajo coeficiente MIUMBC.
Introduction The renewed interest in the 4-quinolones class of antimicrobial agents during the last few years has led to the development of an increasing number of new drugs with very low minimal inhibitory concentrations (MIC) against a variety of bacterial species. The feasibility of achieving adequate serum concentrations with these new drugs made the use of these quinolones possible for treatment of infections other than urinary tract infections. This is an important difference between the modern 4-quinolones and their predecessors, nalidixic acid and other heterocyclic carbonic acid derivatives. Some of these 4-quinolones have already been tested against some mycobacterial species [l-71. Special attention has been given to two drugs with a very wide spectrum: ciprofloxacin [81 and ofloxacin [9]. It has been shown [I] that MlCs of ciprofloxacin were significantly lower than MlCs of norfloxacin in agar dilution tests with M. tuberculosis, M. avium, M. kansasii, M. chelonae, and M. fortuitum. Ofloxacin (DL8280) produced an inhibitory and bactericidal effect against M. tuberculosis in vitro 121 and was found effective in single-drug chemotherapy of 19 patients who were ‘treatment-failure’ cases and had chronic cavitating tuberculosis with multiple drug resistance to the standard antituberculosis drugs [31. The aims of the present study were to determine the MICs, MBCs and MIUMBC ratios of ofloxacin and ciprofloxacin against M. tuberculosis and M. avium complex.
Materials Antimicrobial
and methods
agents
Ciprofloxacin was obtained from Miles Pharmaceuticals, Ofloxacin (Daiichi Pharmaceutical Co.) was obtained from Dr M. Tsukamura. A 1280 yg/ml solution of each drug was made in distilled water for irrigation (Abbott Laboratories) and sterilised by filtration through a Nalgene filter unit (Nalge Company). Serial doubling dilutions of the stock solution were made in sterile distilled water for irrigation, to provide a range of concentrations from 1280 pg/ml to 5 pg/ml. Aliquots of each concentration were stored at -70 “C. Cultures Forty-six strains of the M. avium complex (i.e. M. avium and M. intracellulare) isolated from patients with disseminated infections were used in this study. Smooth transparent colonies from 7Hll agar plates were subcultivated in 7H9 broth. From this point on, references in this paper to M. avium assume M. avium complex. Forty clinical isolates and H& strain of M. tuberculosis were tested. Twenty-three strains had previously been found to be resistant, by the conventional proportion susceptibility test, to at least one antituberculosis drug. Seventeen strains were susceptible to all the antituberculosis drugs. These strains were subcultivated in 7H9 broth. Aliquots of the M. avium and M. tuberculosis broth cultures were kept at -70°C.
Ciprofloxacin M/C
determination
in 7H1.2 broth
by the radiometric
and ofloxacin
269
method
7H12 broth, which contains “C-labelled palmitic acid as a source of carbon, was used. Growth leads to degradation of the palmitic acid and release of 14C02 into the atmosphere above the medium. The BACTEC TB-460 instrument (Johnston Laboratories) detects the amount of 14C02 and records it on a scale of zero to 999 as the Growth Index (GI). Vials were tested daily on the BACTEC instrument and the GI was recorded. Before testing M. avium, an initial vial of 7H12 medium was inoculated with a I:50 dilution of a fresh 7H9 broth culture adjusted to the optical density of a No. 1 McFarland standard. When the GI of the vial reached 999, the broth was diluted 1 :lOO, and 0.1 ml was used to inoculate each of a set of test vials. This inoculum provided a bacterial concentration of IO4 to IO5 CFU-ml (11). lnoculum of M. tuberculosis was prepared by seeding a 7H12 vial with a I:2 dilution of a fresh 7H9 broth culture adjusted to equal a No. 1 McFarland standard. When growth in this vial reached GI 400 to 500,O.l ml of the undiluted broth was inoculated into each of a set of test vials, providing IO4 to IO5 CFU/ml. Two drug-free controls were used with each set of tests for both species. One control was inoculated in the same way as the test vials, the second was inoculated with a 1 :I00 dilution of the inoculum to represent 1 % of the bacterial population. M. avium strains were first tested against 16 pg/ml of each drug. Strains susceptible to this concentration were tested against 8.0, 4.0, 2.0, and 1.0 pg/ml to determine the actual MIC. The narrow range of MlCs against M. tuberculosis made it possible to test 2.0, 1 .O, and 0.5 pg/ml of each drug in a primary titration. The concentration of drug producing a daily GI increase and final GI reading lower than those in the 1 :I00 control was considered to have inhibited more than 99 % of the bacterial population and was therefore defined as the MIC. The tests were carried out for no less than 4 or more than 8 days with M. avium, or no more than 10 days with M. tuberculosis. The final day of observation with M. avium was that on which the GI of the undiluted control reached 999. For M. tuberculosis the endpoint of the experiment came when the GI of the 1 :I00 control was greater than 30 for 2 consecutive days. M/C determination
in 7H12
broth
by plating
The determination of MIC by plating was carried out by the same technique as previously described except that duplicate vials were used. Samples were taken from alternate vials on various days and diluted appropriately, based on the GI reading, so as to have 50-500 CFU per plate. The dilutions were inoculated onto 7Hll ‘agar plates, 0.5 ml per plate, and distributed by tilting the plate. Incubation was at 37 “C in 5 % CO2 for 12-14 days, after which the colonies were counted, The MIC was the lowest concentration that inhibited more than 99 % of the bacterial population in broth for up to 8 days.
M/C determination
by the agar dilution
method
Quadrant petri dishes were prepared with 7Hll agar so as to have a different concentration of drug in each of three quadrants; the fourth quadrant was the drug-free control. Against M. avium strains, concentrations of 0.5-l 6.0 pg/ml of each drug were used while 0.5, 1 .O and 2.0 pg/ml of each drug were tested with M. tuberculosis. Each quadrant was inoculated with 0.1 ml of a bacterial suspension. For M. avium the inoculum was either a lop4 dilution of a 7H12 broth culture that had reached GI 999, or a 1O-5 dilution of a 7H9 broth culture adjusted to the optical density of a No. 1 McFarland standard. For M. tuberculosis the
270
Heifets
and
Lindholm-Levy
inocuium was a IO-* dilution of a 7H12 broth culture that had reached GI 400 to 500. These inocula contained 200-300 CFU. The plates were incubated at 37 “C in the presence of 5 % COz for 12-14 days. The lowest concentration of drug that inhibited more than 99 % of the bacterial population on agar plates was considered to be the MIC. MBC
determination
7H12 vials were inoculated with the organism in the same way as described above for radiometric MIC determination, except that duplicate vials were used and a 1 :I00 control was eliminated. Beginning with an inoculum of IO4 to IO5 CFU/ml, the cultures were allowed to incubate until growth was in the exponential phase and the number of CFU/ml was IO5 to 106. Previous studies [IO-123 showed that for M. avium this bacterial concentration occurred when the GI was approximately 400. At this when the GI was 20-80, and for M. tuberculosis time the drugs were added to achieve 1, 2, 4 and 8 times the previously determined MIC. Samples were taken from alternate vials at various time points, diluted based on the GI reading, and 0.5 ml of the dilutions was inoculated onto 7Hll agar plates as previously described. Incubation was at 37 “C for 12-14 days, after which the colonies were counted and CFU/ml calculated. The MBC was the lowest concentration of drug that killed more than 99.9 % of the bacterial population present when the drugs were added. MIUMBC
ratios
Six strains of M. avium and three strains of M. tuberculosis were tested simultaneously for MIC and MBC of both drugs by the plating methods previously described. The ratio of MIC to MBC was then calculated.
Results Growth Curves in 7H12 broth We confirmed in this study our previous finding [IO, 11, 121 that the maximum available numbers of CFU/ml in 7H12 broth were: for M. tuberculosis IO6 to IO7 CFU/ml, and for M. avium, IO* to IO’ CFU/ml. It is known that the time required to achieve maximum growth in 7H12 broth depends on the inoculum size [13]. In this study, the initial concentration of bacteria in 7H12 broth by the procedure described was between IO4 and IO5 CFU/ml. Under these conditions the maximum growth for most M. tuberculosis strains was achieved within 7-10 days, with the exception of some strains with multiple drug resistance, which produced poor growth in this and any other media. In experiments with M. avium the maximum growth was achieved within 4-8 days. The average generation time during the exponential phase of growth was: for M. tuberculosis strains, 38-48 hours; for M. avium strains, 9-12 hours. Examples of growth curves of M. tuberculosis and M. avium are presented in Figure 1 (curves Al). The exponential phase of growth in drug-free medium (curves Al) was detected in this experiment for M. tuberculosis from the third to the eighth day, and for M. avium from the second to the fifth day of incubation. The daily GI reading curves (Bl in Fig. 1) correlated well with the growth curves (Al) and M. avium. These curves also during the exponential phases for both M. tuberculosis correlated well in the presence of ciprofloxacin (A3 & 83) and ofloxacin (A4 81 B4). These experiments, conducted with three M. tuberculosis and six M. avium strains, confirmed that the inhibition of GI in vials containing ciprofloxacin or ofloxacin reflected actual inhibition of growth by these drugs. These data correspond with previous findings in determining the MlCs for rifabutine. (ansamycin LM427) [I 1 I and ethambutol [I 21. This justified the use of the radiometric method for determination of MlCs of ciprofloxacin and ofloxacin in liquid medium for the remaining 38 strains of M. tuberculosis and 40 strains of M. avium.
Ciprofloxacin
and ofloxacin
271
8 J& tuberculosis
7
A
E
tuberculosis
1 lvllaviYm/_.
2
Days
4
,
6
6
Days
Figure 1. Examples of correlation between the growth curves (CFU per ml) (A) and the daily GI reading curves (6) in experiments for determination of the MlCs of ciprofloxacin and ofloxacin. l-undiluted control; Z-1:100 control; 3-in presence of ciprofloxacin (MIC); 4-in presence of ofloxacin (MIC).
Minimal
inhibitory
concentrations.
MlCs of ciprofloxacin for M. tuberculosis strains were between 0.25 and 2.0 yglml when determined in 7H12 broth radiometrically and 0.125 to 2.0 pg/ml when determined in 7Hll agar plates (Table I). The MlCs of ciprofloxacin for M. avium were significantly higher when determined either by the broth (radiometric) or agar dilution method. Another difference was that the range of MlCs for M. avium was much wider than for M. tuberculosis. For only some of M. avium strains was the MIC as low as the MIC range for M. tuberculosis. No significant differences were found between broth- and agar-determined ciprofloxacin MlCs for M. tuberculosis, but the broth-determined MlCs were lower than agar-determined MlCs for most M. avium strains (Fig. 2, A). The MlCs of ofloxacin for M. tuberculosis were in a narrow range of 0.25 to 2.0 pg/ml when determined in 7H12 broth (Table II), which is the same as for ciprofloxacin. The agardetermined MlCs of ofloxacin were between 0.5 and 1.O yglml. The MlCs of ofloxacin for M. avium strains were much higher than for M. tuberculosis (Table II), and ranged between 4.0 and more than 32.0 pg/ml by either method. The broth- and agar-determined MlCs of ofloxacin had the same value for most of the M. tuberculosis strains; the broth-determined MlCs were slightly lower than agar-determined for about half of the M. avium strains (Fig. 2, B).
272
Heifets
Table I.
and Lindholm-Levy
MlCs of ciprofloxacin Organism
for 41 M. tuberculosis M/C
M. fuberculosis M. avium
fjcg/mlI
in 7Hll
agar
Range
50 %
90
0.125-2.0 1.1X32.0
0.35 8.0
0.48 23.0
M. Aviun -7Hl2 32.0
2
8.0
.
i/
I c
4.0 2.0
-
1.0
m
e
-
.
%
(pg/mll
in 7H72
broth
Range
50
90
0.25-2.0 0.5-16.0
0.34 3.0
0.78 22.0
.
I
%
%
CC@BJX
2.0
4.0
i
/ .
-
.
.
7Hll
AGAR 6.0
16.0
.
.
7Hll
.
1.0
M/C
strains.
A
BROTH
16.0
-i
and 46 M. avium
:/
AGAR
32.0
1.0
2.0
4.0
6.0
16.0
32.0
uglml
lrdml M
tuberculosis 7H12 BROTH
B /
2.0l.O-e s
7Hll AGAR 0.125
0.25
0.5
1.0
2.0
Figure 2. MlCs of ciprofloxacin (A) and ofloxacin 40 M. tuberculosis and 46 M. avium strains.
Minimal
bactericidal
concentrations
t/,1,
4.0
(6) determined
and MWMBC
0.125
0.25
,:“p:” 0.5
1.0
2.0
by 7H12 broth and 7Hll
4.0
agar plate methods
for
ratios
In experiments conducted in 7H12 broth, the MBCs were determined on the basis of comparisons between the kinetics of the number of CFU/ml in drug-free medium and in the presence of different concentrations of drugs added on the 2nd to the 8th day of cultivation when the radiometric readings indicated that the culture was in an exponential phase and the number of CFU/ml was above 105. The actual determination of the number of CFU/ml in broth by plating culture samples, confirmed that the number of CFU/ml when the drug was
Ciprofloxacin Table II.
MlCs of ofloxacin Organism
MIC
M. tuberculosis M. avium
Table
III.
for 41 M. tuberculosis
Bactericidal
in 7till
agar
M/C
Range
50
90
Range
50
0.5-l .o 4.0->32.0
0.5 32.0
0.26-2.0 4.0->32.0
0.7 12.0
activity
Organism
(hg/mll
Table
%
1 .o >32.0
effect
CFlJ/ml when drug added
H3,Rv
%
MBC
By CFlJ/ml
Wml)
6.4~10~ 1.5x105 8.9x IO5
2.0 2.0 2.0
1.0 1 .o 0.5
M. M. M. M. M. M.
3.6~10~ 2.0x 105 4.8x lo5 3.4x lo5 2.0x lo5 1.4x105
4.0 8.0 8.0 16.0 32.0 64.0
4.0 1.0 1 .o 8.0 16.0 16.0
IV.
3350-4 1017-5 4534 3337-4 3349-4 168-4
Bactericidal
activity
Organism
avium avium avium avium avium
M. avium
%
90
%
1.4 28.0
Ratio MIUMBC
Radiometrically 1.0 1.0 0.5 4.0 1.0 1.0 8.0 16.0 16.0
6.4x lo5 l.5X105 8.9x 10s
M/C fug/ml) in the same experiment
effect
CFWml when drug added
M. M. M. M. M.
in 7H1.2 broth
1:2 I:2 1:4 1:1 1:8 1~8 1:2 1:2 I:4
of ofloxacin.
Bactericidal
H3,Rv M. tuberculosis M. tuberculosis#3105 M. tuberculosis#2923
strains.
MIC lpg/ml) in the same experiment
M tuberculosis#3105 M. tuberculosis#2923 avium avium avium avium avium avium
/ug/ml)
273
of ciprofloxacin.
Bactericidal
M. tuberculosis
and 46 M. avium
and ofloxacin
MBC
By CFlJ/ml
Wmll 2.0 2.0 2.0
Ratio MIUMBC
Radiometrically
1 .o 1.0 0.5
1.0 1.0 0.5
I:2 1:2 114
1017-5 453-4 3350-4 3337-4 3349-4
2.0x105 4.8~10~ 3.6x lo5 3.4x 10s 2.0x105
16.0 16.0 64.0 64.0 256.0
4.0 4.0 16.0 16.0 64.0
4.0 4.0 16.0 16.0 64.0
1:4 114 1:4 1:4 1:4
168-4
1.4x105
512.0
32.0
32.0
1:16
added was: for M. tuberculosis, from 1.5x lo5 to 8.9x 1Or’; and for M. avium, from 1.4x IO5 to 4.8~10’ (Tables III and IV). To have the most accurate MIUMBC ratios, the MlCs were determined by two methods with the same strains: by sampling and plating and by radiometric GI readings as previously described. Unlike the MBC determination, the drugs were added at the beginning of cultivation, when the concentration of bacteria was between
274
Heifets
and
Lindholm-Levy
IO4 and IO5 CFU/ml. In experiments with three strains of M. tuberculosis and six strains of IM. avium we found no difference in the MIC values determined either by sampling (CFU/ml] or GI radiometric readings (Tables III and IV]. The MBC of ciprofloxacin (Table Ill] was 2.0 pg/ml for all three strains of N1. tuberculosis, and between 4.0 and 64.0 ug/ml for six M. avium strains. The low MIC/MBC ratios indicated high bactericidal activity: 1:2-l :4 for M. tuberculosis, and 1:2-I:8 for M. avium. The MBCs of ofloxacin (Table IV) were the same as those of ciprofloxacin (2.0 mg/ml) for M. tuberculosis strains, but were substantially higher for M. avium strains (from 16.0 to 512.0 kg/ml). The MIC/MBC ratios were I:2 to I:4 for M. tuberculosis, and I:4 for five of six strains of M. avium. Discussion The mean serum peak level (C,,,) of ciprofloxacin is between 2.0 and 3.0 pg/ml attainable 1.25 h after an oral administration of 500 mg [14-171 with good penetration into different tissues [14, 16, 171. The concentration in sputum from patients with cystic fibrosis is 1 .Ol f0.24 pg/ml [181. The peak levels in serum are higher after a 750 mg oral dose [19]. The pharmacokinetics of ofloxacin have similar features, with a peak serum level of 10.7 pg/ml after administration of 600 mg orally [20]. The MlCs of both ciprofloxacin and ofloxacin for M. tuberculosis were much lower than the achievable serum and tissue levels per site. The MlCs of ciprofloxacin for 90 % of M. tuberculosis strains (MIC& were 0.48 pg/ml or less when determined in 7H12 broth. These data correspond with previous findings: 1 pg/ml [I] and 0.5 pg/ml [5] when the MIC was determined in 7HlO agar plates. The MI&, of ofloxacin against M. tuberculosis in this study was less than 1 .O yg/ml when determined in 7Hll agar plates, and 1.4 yglml in 7H12 broth, with a range of 0.5-1.0 and 0.25-2.0 ug/ml, respectively. These data also correspond to findings by other authors: 0.67-1.25 yglml in Ogawa medium [2], and 0.5-1.0 pg/ml in 7HlO agar plates 151. Comparisons of in vitro activities of the two quinolones in this, as well as in a previous study [51, indicate that the MlCs of ciprofloxacin were lower than MlCs of ofloxacin (see also Fig. 2). The MlCs of ciprofloxacin and ofloxacin for M. avium complex strains were substantially higher than for M. tuberculosis, and were in a wide range, regardless of the method of determination (in 7Hll agar plates or in 7H12 broth). For only 13 of 46 M. avium strains were the broth determined MlCs of ciprofloxacin within the same range as for M. tuberculosis (2.0 pg/ml or less). There were no strains of M. avium for which the MlCs of ofloxacin were within this range. Ciprofloxacin is probably more active than ofloxacin against M. avium. In general, both drugs are less promising against M. avium than against M. tuberculosis, because the MlCs against the former were, for many strains, higher than the achievable serum concentrations. At the same time, the following two facts should be taken into account: concentrations of ciprofloxacin in macrophages are two to three times higher than in the extracellular fluid [21], and ciprofloxacin has a wide range of MlCs against M. avium. The broth-determined MlCs of ciprofloxacin for more than half of M. avium strains (28 of 46) were 4.0 pg/ml or less, lower than the concentrations achievable within the macrophages. This fact indicates that ciprofloxacin may be effective in treatment of those patients whose M. avium isolate is susceptible to low concentrations of the drug and that an MIC should be determined against each strain, preferably in broth, which is a more sensitive technique, providing more accurate information. The perspective of practical application of ciprofloxacin may be broadened should the synergistic or additive effect shown with other drugs apply as well to ciprofloxacin, lowering its MIC against M. avium strains at least twofold.
Ciprofloxacin
and ofloxacin
275
Another important feature of ofloxacin and ciprofloxacin, confirmed in this study, is the high bactercidal activity of these drugs, with MIUMBC ratios of I:2 to 1:4 for both drugs against M. tuberculosis, 1:1-l :8 for ciprofloxacin, and I:4 for ofloxacin against M. avium.
Acknowledgements This work was supported by contract No. l-Al042544 with the National Institute of Allergy and Infectious Disease. We thank Dr M. Tsukarmura for providing us with ofloxacin for this work, Miles Laboratories for supplying us with ciprofloxacin, B. Silverstein for the artwork, and C. J. Queen for preparation of the manuscript.
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DeYoung, D. R., Roberts, G. D. (1984). In vitro activities of norfloxacin and CiprOfkOXaCin against Mycobacrerium tuberculosis, M. avium complex, M. chelonei, M. fort&urn, and M. kansasii. Antimicrobial
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and
Lindholm-Levy
19 Gonzalez, M. A., Uribe, F., Moisen, S. 0.. Fuster, A. P., Selen, A., Welling, P. G., Painter, 6. (1984). Multiple-dose pharmacokinetics and safety of ciprofloxacin in normal volunteers. Antimicrobial Agents and Chemotherapy, 26, 741. 20 Lockley M. FL, Wise, R., Dent, J. (1984). The pharmacokinetics and tissue penetration of ofloxacin. Journal of Antimicrobial Chemotherapy, 14, 647. 21 Easmon, C. S. F., Crane, J. P. (1985). Uptake of ciprofloxacin by macrophages. Journal of Clinical Pathology, 38, 442.