as well as those of newly described drugs clarithromycin (a macrolide) and sparfloxacin (a difluoroquinolone), were assessed against three strains of the ...
CURRENT MICROBIOLOGY Vol. 26 (1993), pp. 191-196
Current Microbiology 9 Springer-Verlag New York Inc. 1993
Ethambutol Potentiates Extracellular and Intracellular Activities of Clarithromycin, Sparfloxacin, Amikacin, and Rifampin Against Mycobacterium avium Nalin Rastogi, Val6rie Labrousse, and Joao Paulo Carvalho de Sousa Tuberculosis and Mycobacteria Unit, Institut Pasteur, Paris, France
Abstract. Intracellular bactericidal activities of the antituberculosis drugs rifampin and amikacin,
as well as those of newly described drugs clarithromycin (a macrolide) and sparfloxacin (a difluoroquinolone), were assessed against three strains of the Mycobacterium avium complex (MAC) growing in two different in vitro macrophage systems, namely, mouse bone marrowderived macrophages (BMM~) and human peripheral blood monocyte-derived macrophages (human M~). All the infected macrophages were fed reported Cma• concentrations of the drugs, i.e., 15/zg/ml for rifampin, 20 tzg/ml for amikacin, 4/xg/ml for clarithromycin, and 1.5/~g/ml for sparfloxacin. Further potentiation of drug activity in the presence of Cmaxlevel of ethambutol (6/zg/ml) during 9 days of intracellular growth (measured by lysing the macrophages and making bacterial counts) was assessed. Our results showed that all four drugs were active against the strains used in this study and that the addition of ethambutol (which had no significant intracellular activity against the bacteria in this system) further potentiated the bactericidal effect of the drugs. When the same drug combinations were tested at their sublethal concentrations by BACTEC | radiometric methodology, a good correlation between the drug enhancement data in extracellular and intracellular systems was found. We conclude that ethambutol may serve as an essential component in effective anti-M, avium chemotherapy and that the effective drug combinations may be routinely screened by the Bactec radiometric methodology.
The multiple-drug-resistant Mycobacterium avium complex (MAC) organisms are opportunistic human pathogens, now frequently associated with AIDS as opportunistic infections [1, 4, 24]. Despite their multiple drug resistance, which has been attributed to an exclusion barrier located in their cell envelope [17], both amikacin [6] and rifampin [18] have been found to be more active against them than other antimycobacterial drugs such as isoniazid or pyrazinamide, which are completely ineffective [18]. Recently a macrolide drug, clarithromycin [16], and a new difluoroquinolone drug, sparfloxacin [20], were found to have high anti-MAC activities. As MAC are intracellular pathogens [3, 14, 15], we decided to compare the intracellular activities of amikacin and rifampin with those of clarithromycin and sparfloxacin, with both murine and human macrophages. In accordance with our previous macrophage studies [18], the infected macrophages were fed Cma• levels
of the drugs. Further potentiation of the intracellular drug activity by ethambutol, which decreases the MAC cell wall barrier by disrupting the wall outer layer [19], by inhibiting both the biosynthesis of arabinogalactan [21] and the transfer of mycolic acids in the mycobacterial cell envelope [22], was also investigated. The drug combinations were also tested at their sublethal concentrations by BACTEC | radiometric methodology to correlate the drug enhancement results obtained with extracellular and intracellular systems. Materials and Methods Bacteria and growth. Mycobacterium
avium strain CIPT 140310003 (serotype 1) and two clinical isolates (90-0827 from an HIV § patient, and 90-1257 from an HIV patient) from the Pasteur Culture Collection were used in this investigation. The bacteria were grown at 37~ in complete 7H9 broth (Difco Laboratories, Detroit, Michigan) containing 0.05% (v/v) of Tween 80 to
Address reprint requests to: Dr. Nalin Rastogi, Unit6 de la Tuberculose et des Mycobact6ries, Institut Pasteur, 25 Rue du Dr. Roux, 75724-Paris C6dex 15, France.
192 avoid clumping, and were harvested at their midlogarithmic phase at an optical density of 0.15 (measured at 650 nm with a Coleman Junior II spectrophotometer), which corresponded to about 108 colony-forming units (CFU)/ml.
Extracellular drug activity. Radiometric determination of MIC against MAC organisms was performed as reported earlier [16, 20]. Briefly, growth of bacteria was monitored in a confined atmosphere with the BACTEC | 460-TB apparatus (Becton Dickinson, Towson, Maryland) by measuring the I4CO2 released during metabolism of ~4C-palmitic acid in 7H12 broth. Growth expressed as a numerical value, the growth index (GI), ranged from 1 to 999. Inoculum for the BACTEC | vials was prepared as follows: after growth of a strain in an initial BACTEC | vial to a GI of 500, the culture was diluted 1 : 10 and 1 : 1000-fold, and 0.1 ml of the 1 : 10 diluted culture was used to inoculate the drug-containing vials as well as a 1 : 10 control (corresponding to about 5 x 103 to 6 • 104 CFU/ml depending on MAC strains studied). The daily GI in the drug-containing vials was compared with another control vial inoculated with 0.1 ml of the 1 : 1000 diluted culture, and the results were interpreted once the GI in the 1 : 1000 diluted control reached a value of 30 or more [16, 20]. MIC was defined as the minimal concentration of drug which resulted in a AGI in the drug-containing sample lower than that in the 1 : 1000 diluted control. For combined action studies, all the drugs were used at sublethal concentrations, i.e., 0.25 p,g/ml for sparfioxacin and 1 /~g/ml for the others. The reason for this choice was the fact that at this concentration the drugs used alone were unable to significantly reduce the initial inoculum added in the BACTEC | vials. In such a case, any significant drug enhancement observed according to the radiometric " x / y " quotient criterion [16, 19, 20] could eventually suggest a reproducible effect in infected host cells, where the drugs are available in much higher concentrations. Briefly, the combined drug action was equal to " x / y " , where " x " was the BACTEC | GI obtained with the combination of two drugs, and " y " was the lowest GI obtained at the same time for any of the drugs used alone. In case of a two-drug combination, an " x / y " value of 1 indicated that there was no interaction between the two drugs, an " x / y " quotient of 2 indicated the presence of antagonism between the two. Macrophage culture. Human macrophages (6-7 days old) from adherent peripheral blood monocytes of healthy blood donors and mouse bone marrow-derived macrophages (BMM0) from Bcg ~, 6- to 13-week-old female C57BI/6 mice were prepared as described recently [20J. The RPMI-1640 growth medium was supplemented with 3% (v/v) heat-inactivated fetal calf serum (FCS) and 2 mM L-glutamine in the case of human macrophages, and with 10% FCS, 2 mM L-glutamine and 10% (v/v) of L-cellconditioned medium in the case of BMM0. Macrophage monolayers containing about 1 x 106 cells/well in 12-well tissue culture clusters (Costar, Cambridge, Massachusetts) and maintained in the presence of 5% CO2 in a water-jacketed incubator at 37~ were used in all experiments. Under our experimental conditions more than 97% of cells were viable as verified by the trypan blue dye exclusion test.
Infection of macrophage monolayers. Macrophage monolayers were infected with bacteria as follows. The bacterial cultures were grown to their exponential phase of growth (to an OD650 of 0.15, corresponding to about 108 CFU/ml) and 0.1 ml of the
CURRENT MICROBIOLOGY Vol. 26 (1993)
culture was added to 10 ml of macrophage culture medium. For infection, 1 ml of the culture medium was removed from each well and replaced with 1 ml of the above medium. Each well containing 106 macrophages was thus inoculated with approximately 106 CFU. The macrophages were allowed to phagocytize bacteria for 4 h at 37~ after which all the extracellular bacilli were thoroughly washed away with Hanks balanced salt solution, and the number of bacteria effectively pbagocytized was determined by lysing the macrophages with 0.25% (w/v) sodium dodecyl sulfate (SDS), doing immediate serial dilutions, and plating the lysates on 7H 11 agar medium for viable count determinations. Addition of 0.25% SDS to parallel cultures of the MAC organisms, which were immediately serially diluted for viability assessment in parallel control experiments, showed that it did not lower the bacterial viable counts. After phagocytosis, macrophage cultures were refed fresh medium containing the antibiotic being tested and were incubated for 2, 4, 7, and 9 days. At each time period macrophages were lysed to release intracellular mycobacteria, which were enumerated on 7Hll agar medium. Results were expressed as mean viable counts -+ standard error/well.
Drugs. In accordance with our experimental model for determining intracellular action [16, 18, 20], all the drugs were used at their reported Cmax in humans, i.e., 20 p,g/ml for amikacin [2], 1.5 /~g/ml for sparfloxacin [12, 13], 4/~g/ml for clarithromycin [ 10], and 15 and 6/~g/ml for rifampin and ethambutol respectively [11]. Sparfloxacin (Rh6ne-D.P.C. Europe, Antony, France), clarithromycin (Abbott Laboratories, North Chicago, Illinois), amikacin (Bristol, Paris, France), and ethambutol (Lederle, Oullins, France) were kindly provided by their manufacturers, whereas rifampin was purchased from Sigma Chemical Co. (St. Louis, Missouri).
Results and Discussion Mycobacteriosis due to MAC has become the most common bacterial infection in AIDS patients in the U.S.A. and Europe, with between 17 and 24% of all the AIDS patients affected [4]. As many as 50% of AIDS patients in some studies have been found to be infected with acid-fast bacilli at post mortem [9]. It has been recently suggested that MAC/HIV dually infected patients had significantly shorter survival than comparable AIDS patients without MAC infections [1, 5, 7]. Although MAC infections do not respond as well as M. tuberculosis infections to standard antimycobacterial chemotherapy, Horsburgh et al. [5] have recently shown significantly longer survival of MAC/HIV-infected patients who received three or more antimycobacterial drugs for a period of at least 3 months. The multiple drug resistance of MAC [which has been attributed to an exclusion barrier located in their cell envelope; [17], their intracellular growth [3, 14, 15], as well as the lack of data on correlation between in vitro drug susceptibility data and the in
193
N. Rastogi et al.: Enhanced Drug Activity Against M, avium
Table 1. MICs of various drugs against the MAC strains measured by BACTEC | methodology MIC (/xg/ml) Strain CIPT 14031003 Clinical isolate 90-0827 (HIV +) Clinical isolate 90-1257 (HIV-)
Rifampin
Amikacin
Sparfloxacin
Clarithromycin
Ethambutol
2 2
1 4
0.5 0.5
2 2
2 2
2
4
0.5
4
2
Table 2. Enhancement by ethambutol of drug activity against extracellularly growing MAC organisms as assessed by radiometric " x / y " quotient calculations Enhancement of drug activity by ethambutoP Strain CIPT 14031003 Clinical isolate 90-0827 (HIV § Clinical isolate 90-1257 (HIV-)
Rifampin
Amikacin
Sparfloxacin
Clarithromycin
+ +++
+ +++
++ +++
+ +++
+
+++
+
+
a All the drugs were used at their sublethal concentrations, i.e., 0.25 txg/ml for sparfloxacin and 1/xg/ml for all other drugs. Enhancement of drug activity was calculated by " x / y " quotients radiometrically as described in text. " x " was the BACTEC | GI obtained with the combination of drugs, and " y " was the minimal GI value for any of the drugs used alone. An " x / y " quotient of ,
$ ~
4
Experimental Conditions
M. avium Strain 90-1257/BMMO 10 8 107
o
9 9 []
--
108]1~
O
O
t
Experimental Conditions
9 Day2
1071 c-
107 10 6
M. avium ClPT and 90-1257 9 Day 2 (CIPT) strains/Human M ~
9 Day 2 9 Day 4 [ ] Day 7 [ ] Day9
~[~
106 iI1~,=,_.,. I]1 . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 s 10 4 10 3
~
u_
EL
~
Experimental Conditions
~
Fig. 1. Intracellular bactericidal effect ofrifampin (RIF), amikacin (AMIK), sparfloxacin (SPAR), and clarithromycin (CLA) used alone and in association with ethambutol (EMB) against various MAC strains growing inside murine bone marrow-derived macrophages. All the drugs were used at their Cmax levels. The data are represented as viable counts/106 MO/well. The initial inoculum is shown by a dotted base line in each figure and was equal to 2.16 -+ 0.26 • 106 in the case of the CIPT strain (A), 1.34 -+ 0.14 x 106 in the case of the clinical isolate 90-0827 from an HIV + patient (B), and 1.1 + 0.16 • 106 in the case of the clinical isolate 901257 from an HIV- patient (C).
exclusion barrier, thus gaining access to the targets in the bacterial cell. The bacterial viable count data showing the effective number of bacteria phagocytized and their intracellular multiplication are represented in Figs. 1 and 2, and the potentiation of drug activity by
,,=,
==
Fig. 2. Intracellular bactericidal effect ofrifampin (RIF), amikacin (AMIK), sparftoxacin (SPAR), and clarithromycin (CLA) used alone and in association with ethambutol (EMB) against various MAC strains growing inside human macrophages. All the drugs were used at their Cmax levels. The data are represented as viable counts/106 M0/well. The initial inoculum is shown by a dotted base line in each figure, and was equal to 2.92 -+ 0.56 x 105 and 1.16 -+ 0.38 x 105 respectively in the case of the CIPT strain and the clinical isolate 90-1257 from an HIV patient (A), and 1.48 + 0.21 x 106 in the case of the clinical isolate 90-0827 from an HIV + patient (B).
ethambutol is summarized in Table 3. In the figures, the initial number of bacteria phagocytized has been represented by a dotted base line, and any value below this line depicts a bactericidal activity. As can be seen from our data, all the drugs used at their Cma• concentrations (except ethambutol) were significantly bactericidal against intracellularly growing MAC organisms. In accordance with our previous observations [8], in general both the bacterial growth and the bactericidal effect of the drugs were more pronounced in human macrophages. Most interestingly, ethambutol, which had only a minimal bacteriostatic effect on its own, was able to enhance the activity of other drugs (Figs. 1, 2, and Table 3). Despite overall agreement between the extracellular and intracellular drug enhancement data, the drug enhancement profiles observed extracellularly
N. Rastogi et al.: Enhanced Drug Activity Against M. avium
195
Table 3. Enhancement of the intracellular activity of drugs against MAC strains growing in murine and human macrophages by ethambutol Murine macrophages MAC strain
Human macrophages
Drug a
day 2
day 9
day 2
day 9
CIPT 14031003
Rifampin Amikacin Sparfloxacin Clarithromycin
0b 0 40.5 0
51.3 58.8 11.7 10.0
11.5 14.2 26.0 26.0
32.0 28.6 89.4 73.0
Clinical isolate 90-0827 (HIV §
Rifampin Amikacin Sparfloxacin Clarithromycin
0 0 46.0 0
8.2 2.0 57.0 18.8
32.0 70.0 31.5 0
61.0 86.8 50.0 50.0
Clinical isolate 90-1257 ( H I V )
Rifampin Amikacin Sparfloxacin Clarithromycin
0 20.7 0 50.8
0 95.3 64.3 33.6
51.7 27.1 28.7 39.4
60.0 50.9 85.7 49.1
a All the drugs were used at their reported Cmax concentrations, i.e., 15 /zg/ml for rifampin, 20/zg/ml for amikacin, 1.5 p~g/ml for sparftoxacin, and 4/zg/ml for clarithromycin. Ethambutol was used at 6/zg/ml in combination with other drugs to assess its ability to enhance their bactericidal effect. b The enhancement of drug action (provided that ethambutol used alone was not bactericidal) was calculated by the equation: viable counts/106 M 0 with drug + ethambutol 1 - viable counts/106 MO with the drug used alone
(Table 2) and intracellularly (Table 3) did not match perfectly, e.g., variations in the degree of enhancement in the case of intracellularly growing bacteria were observed not only according to the MAC strains used, but also depending on the macrophage systems used (murine or human). In general, overall bactericidal activity of the drugs (Figs. 1 and 2), as well as their enhancement by ethambutol (Table 3), was higher in human macrophages. These results are in agreement with our previously published data [20]. Apart from major differences in the drug susceptibility profiles of various MAC strains [16, 20], the fact that drug combinations could be less or more bactericidal against the same MAC strain growing in different macrophage systems is clear evidence that factors depending on intricate bacteria-macrophage interactions (as reviewed in [15]; e.g., phagosome-lysosome fusion inhibition, inhibition of fusion of incoming pinosomes with other intracellular compartments, perturbation of membrane flux, etc.) may further change the final outcome of antimicrobial therapy of these intracellular pathogens. In conclusion, present data show that extracellular enhancement of antimycobacterial drugs by ethambutol used at sublethal concentrations, as evidenced by BACTEC | radiometry in in vitro assays, was successfully reproduced when these drug com-
x
100
binations were assayed at much higher Cmax levels against MAC strains growing in both murine and human macrophages. In addition, we also found that this enhanced bactericidal effect was evidenced both in clinical isolates from H I V - or HIV + individuals. These results confirm published data [16, 19, 20] and support earlier propositions about the synergistic role played by ethambutol with other antimycobacterial drugs [8, 23]. We conclude that despite a lack of any bactericidal effect per se against MAC organisms when used alone, ethambutol does potentiate the intracellular bactericidal activity of other drugs. Ethambutol therefore should be regarded as an essential component in the effective anti-MAC chemotherapy, and routine screening of combined drugs by radiometric methodology may help the clinicians to make a better (if not yet perfect) choice to tackle otherwise difficult-to-treat MAC infections. However, whether intricate bacteria-macrophage interactions could further modify the outcome of antiM. avium therapy must now be further studied.
ACKNOWLEDGMENTS We thank J.-P. Chauvin (Abbott-France) and H. Lecoeur (Rh6neD.P.C. Europe) for kindly providing clarithromycin and sparfloxacin respectively; B. Quiviger (Becton Dickinson-France) for
196
CURRENTMICROBIOLOGYVol. 26 (1993)
providing the Bactec | 460-TB apparatus and growth medium; and B.B. Vargaftig and M. Bachelet (Institut Pasteur) for their help in obtaining human macrophages. 12.
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