a Unité de la Tuberculose et des Mycobactéries, Institut Pasteur, Morne Jolivière BP 484, 97165 Pointe à Pitre Cedex, Guadeloupe b Domaine Antibiothérapie, Hoechst Marion Roussel, 93230 Romainville, France c Laboratoire de Microbiologie, Centre Hospitalier Victor Dupouy, 95107 Argenteuil Cedex, France
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Abstract |
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Introduction |
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Materials and methods |
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We studied 25 isolates of the M. tuberculosis complex (see Table), 10 of which were M. tuberculosis (seven rifampicin-susceptible isolates and three rifampicin-resistant isolates, as determined by the 1% proportional method using 7H11 agar with a critical rifampicin concentration of 1 mg/L), five M. africanum, five M. bovis and five M. bovis BCG. All strains were from our own culture collection and were freshly cultured on LöwensteinJensen medium before the experiments.
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Bacterial growth was monitored in a confined atmosphere using the Bactec 460-TB apparatus (Becton Dickinson, Sparks, MD, USA); this determines the ability of bacteria to catabolize [14C]palmitic acid in 7H12 broth by measuring the 14CO2 released. The growth of the bacteria is represented as a numerical value called growth index (GI), which ranges from 1 to 999. The initial bacterial inoculum was standardized as reported previously for M. tuberculosis.5,10,11 Strains were first grown in a Bactec vial to a GI of 500, then drug-containing vials were inoculated with 0.1 mL of this culture. The change in daily growth index (GI) of the drug-containing vials was compared with that in a control vial which was initially inoculated with 100 times fewer bacteria in the absence of drug (1/100 control). Under these conditions, the MIC was interpreted once the GI in the 1/100 control reached a value of
30 and was defined as the lowest drug concentration for which the
GI was less in the drug-containing sample than in the control.5,10,11
In addition to MICs, MBCs were also determined for selected strains using a method reported previously for M. tuberculosis.10 The MBC was defined as the lowest concentration of drug that effectively reduced the bacterial viable counts in the drug-containing sample as compared with the initial inoculum by 99%. For this purpose, the cfu/mL for each strain was determined (i) at the time of inoculation of Bactec vials (time 0) and (ii) at the end of the experiment, as follows: 0.1 mL of culture from the Bactec vials was removed and serial 10-fold dilutions in sterile double-distilled water were prepared, giving 10-, 102-, 103- and 104-fold dilutions and 0.1 mL of each of these dilutions was plated on to 7H11 agar medium. The resulting bacteria were counted after 21 days of incubation at 37°C. This procedure avoided the possibility of accidental reduction in bacterial viability as a result of carryover of drugs to the solid medium.10,11
MIC determination using Middlebrook 7H11 agar medium
For this purpose, the 1% proportional method11 was used.11 Briefly, bacteria were scraped from LöwensteinJensen slants and thoroughly homogenized in sterile distilled water with 2 mm glass beads. The suspension was allowed to stand for about 2 min to remove aggregates of organisms; the upper portion of the suspension was carefully removed and the optical density at 650 nm was adjusted to 0.15. Bacterial suspensions were diluted stepwise up to 105-fold dilutions, and 1 mL of 102-, 104- and 105-fold dilutions were plated on to 7H11 agar medium containing the desired concentrations of the drugs to be tested. The plates were incubated at 37°C and the resulting bacterial counts were determined after 21 days of incubation. The MIC was defined as the lowest drug concentration that inhibited >99% of bacterial colonies as compared with the counts for control untreated bacteria.11
Drugs
Rifapentine, 25-O-desacetylrifapentine and rifampicin were provided by Hoechst-Marion-Roussel, Romainville, France, and rifabutin was obtained from Pharmacia, Guyancourt, France. All stock solutions were initially prepared in dimethylsulphoxide (DMSO) and serially diluted in sterile distilled water before use. Equivalent amounts of DMSO did not inhibit the growth of control bacteria treated likewise.
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Results |
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It is interesting to note that the activity of all the rifamycins tested was highest against M. bovis BCG (MIC range 0.0080.063 mg/L for rifapentine and 0.0160.125 mg/L for its metabolite). For this species, MICs of rifapentine were either comparable to that of rifabutin or one dilution lower, and the MICs of 25-O-desacetylrifapentine were either comparable to that of rifampicin or one dilution lower (Table). MICs determined using the 1% proportional method on 7H11 agar medium were not always one or two dilutions higher for M. bovis BCG, unlike most other species (Table
).
The three isolates of M. tuberculosis with a high degree of resistance to rifampicin (MICs >32.0>64.0 mg/L) were also resistant to all the drugs tested, including rifapentine; however, the MICs of rifabutin in this case were lower than that of rifapentine (Figure 1, Table
).
The bactericidal effect of the four rifamycins against the isolates tested are compared in Figure 2. The results obtained for fixed concentrations (0.125, 0.25 and 0.5 mg/L) of each drug against representative drug-sensitive isolates of M. tuberculosis (Figure 2a
), M. africanum (Figure 2b
) and M. bovis and M. bovis BCG (Figure 2c
) show that the bactericidal activity of the 25-desacetyl metabolite was comparable to that of rifampicin, and that rifapentine used at the same concentration was significantly more bactericidal than rifampicin (it resulted in a 2-log higher killing at 0.5 mg/L). For a significant proportion of drug-sensitive isolates, rifapentine was also more bactericidal than rifabutin, with a 1-log higher killing effect (Figure 2
). In contrast, none of the rifamycins used showed remarkable bactericidal activity against rifampicin-resistant M. tuberculosis clinical isolates (Figure 2d
). In conclusion, irrespective of the method used, the rifapentine MICs for all rifampicin-susceptible isolates of M. tuberculosis complex (M. tuberculosis, M. africanum, M. bovis and M. bovis BCG) were
0.5 mg/L, whereas resistance to rifamycins was always associated with much higher MICs (Table
). We suggest, therefore, that a rifapentine breakpoint concentration of 1.0 mg/L is suitable for distinguishing drug-susceptible and drug-resistant clinical isolates by the Bactec radiometric method or the 1% proportional method using 7H11 agar. However, a better correlation of rifapentine and rifampicin MICs would require studies on a number of M. tuberculosis isolates with varying levels of rifampicin susceptibility.
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Discussion |
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In this context, the recent approval by the United States Food and Drug Administration of rifapentine, a cyclopentyl-substituted rifamycin, has made this the first new antituberculosis drug to be licensed in 25 years.16 After the intensive first phase of the short-course chemotherapy, when rifapentine is administered twice weekly for 2 months, just one dose of rifapentine once a week is reportedly sufficient for the next 4 months instead of the present twice-weekly dose of rifampicin.7,16 This is expected to increase compliance, thereby decreasing the probability of the emergence of secondary resistance to rifamycins in general.16
The MICs of rifapentine and its 25-desacetyl metabolite against drug-susceptible isolates of M. tuberculosis, M. africanum, M. bovis and M. bovis BCG (Table) are easily achievable in humans (the Cmax of rifapentine and 25-O-desacetylrifapentine are about 15 mg/L and 46 mg/L, respectively, after oral administration of a single 600 mg oral dose). At a concentration of 0.5 mg/L, rifapentine results in a c.24 log reduction in initial bacterial inoculum, and its 25-desacetyl derivative can reduce the initial bacterial inoculum by 12 logs at the same concentration (Figure 2
), so the achievable serum concentrations of these two compounds represent a Cmax/MBC ratio of about 10 for 25-O-desacetylrifapentine and of
30 for rifapentine.
We also compared the results obtained with the reference compound rifampicin and a more recent drug, rifabutin. Rifabutin, a spiro-piperidyl rifamycin S compound previously known as LM 427, shows considerably greater in vitro activity than rifampicin against both M. tuberculosis and Mycobacterium avium,1719 and has recently been used successfully to treat patients with newly diagnosed pulmonary tuberculosis.20,21 Like rifapentine, rifabutin has a prolonged half-life.17 These two agents have comparable activity against M. tuberculosis (reference 7 and results of this study). However, the achievable serum concentration of rifabutin, 0.34 mg/L, allows a considerably lower Cmax/MBC ratio (12) than for rifapentine (about 30) and 25-O-desacetylrifapentine (10) (see above). Consequently, the in vitro antituberculosis activity of rifapentine (reference 5, 7 and 19, and this study), its pharmacokinetic properties13,5 and its recently described efficacy in treating tuberculosis in models of experimental infection and in human clinical trials alike79,16 argue in favour of its use as a first-line antituberculosis drug.
However, the emergence of acquired resistance to rifabutin during unsuccessful chemotherapy of a patient with a rifampicin-containing regimen,22 to rifamycins in patients treated with rifapentine23 and to rifampicin in patients receiving rifabutin prophylaxis24 calls for the utmost care and vigilance while using the newer rifamycins. Their higher activity can neither compensate for the emergence of acquired resistance in patients as a result of lack of compliance, nor avoid the emergence of cross-resistance to new generations of rifamycins in patients infected with resistant strains of tubercle bacilli that are unknowingly subjected to a standard short-course chemotherapy regimen for long periods because of a delayed diagnosis of drug resistance.
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Notes |
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References |
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Received 4 January 2000; returned 23 March 2000; revised 12 April 2000; accepted 22 June 2000