The antimicrobial treatment of MAC infections, particularly in patients with AIDS, is difficult. This is in part due to the severely depressed state of host defense mechanisms in these patients, resulting in reduced macrophage antimicrobial capacity (4
). In addition, the variability of MAC isolates in susceptibility to most antimycobacterial agents, except for macrolides, is high (29
A number of agents have been suggested for treatment of MAC infections (3
). Mostly macrolide-containing multidrug treatment regimens are applied (3
). In clinical studies the macrolide CLR showed high efficacy against disseminated MAC infections in patients with AIDS or non-AIDS patients with localized pulmonary disease (2
). The macrolides (particularly CLR) are the only antimicrobial agents for which a correlation between in vitro susceptibility tests (broth dilution) for MAC and clinical response has been demonstrated in controlled clinical trials (25
). Empirical regimens in the treatment of MAC infections are recommended (3
As clinical studies show a high risk for relapse and emergence of resistant isolates during monotherapy with macrolides, combinations of agents are used primarily in order to reduce the incidence of macrolide resistance. However, uncertainty persists regarding the optimal drugs that should accompany a macrolide (6
). EMB is recommended as a second drug (31
). It has been shown in vitro (34
) as well as in vivo in a mouse model of disseminated MAC infection (8
) that the combination of CLR and EMB is effective at reducing the incidence of CLR resistant mutants. However, the addition of EMB appeared to have no significant effect on the reduction of bacterial numbers (19
). Also, a clinical study of Dube et al. shows that the addition of EMB to a CLR regimen results in a reduction in the development of resistance but does not enhance clearance rates of MAC from the blood (15
Rifamycins, including RIF, have the best potential as a third agent, although their role is not fully clarified. In addition, RIF, by inducing the hepatic cytochrome P450 pathways, results in substantial reductions in the bioavailability of CLR (37
). For severe cases of MAC disease, other antimycobacterial drugs such as quinolones and aminoglycosides are suggested. The potential of fluoroquinolones or aminoglycosides is of considerable interest because of the proven clinical activity of these agents against other mycobacterial species, including Mycobacterium tuberculosis
Quinolones including CIP, SPX, and MXF, have been used in relatively effective MAC multidrug treatment regimens (7
). MXF particularly had significant anti-MAC activity in beige mice (7
). Still, the antimicrobial activity of each of the quinolones is not yet fully understood. The aminoglycosides AMK and STR are highly active and reduce the incidence of resistance in macrolide-containing regimens in animal models of MAC (33
). Aminoglycosides have been used in clinical studies in successful treatment regimens, whereas other investigators failed to show a beneficial effect of aminoglycosides (36
). However, Fattorini et al. demonstrated in in vitro assays that AMK significantly improved the activity of the CLR-EMB combination (17
). The potential risks and benefits of treatment with aminoglycosides should be weighed carefully. These agents should be considered especially as part of short-term induction therapy in view of toxicity observed with long-term administration.
Although the macrolides are recognized to be the most active of the drugs available for MAC treatment, important questions remain in defining optimal treatment. The relative contribution in terms of antimicrobial activity of each of the components in the multidrug regimen is not clear. Some patients receiving multidrug macrolide-containing regimens who initially respond to therapy develop relapse of infection, which is often the result of insufficient antimicrobial activity (assuming adherence to the drug regimen and no further decline of the host's cellular immune response).
The MAC strain 101 used in the present study, originally isolated from the blood of an AIDS patient with disseminated MAC infection, appeared CLR susceptible following the breakpoints for determining susceptibility and resistance according to the NCCLS guidelines (35
). The data show that five relevant antimycobacterial agents, CLR, EMB, RIF, AMK, and MXF, all inhibited mycobacterial growth at clinically relevant concentrations but differed with respect to their bactericidal activity during 21 days of exposure of the early-log-phase MAC. At the end of the 21-day incubation period, compared to the concentrations resulting in growth inhibition, the concentrations needed to achieve a bactericidal effect (≥99% killing) were always twofold or fourfold higher. CLR, RIF, and AMK particularly showed concentration-dependent killing.
However, with respect to the bacterial killing rate, the agents differed markedly. For AMK the high bactericidal activity (≥99% killing) observed after 21 days was already obtained after 3 days of exposure. In contrast, for MXF the concentration needed to achieve bactericidal activity within 3 days was 16-fold higher compared to the concentration needed at 21 days of exposure. At the end of the incubation period of 21 days MXF was superior over AMK, whereas after 3 days of incubation AMK appeared superior over MXF. Actually, the high killing rate of AMK is unique, and in this respect AMK is superior to MXF as well as CLR, EMB, and RIF. RIF showed concentration-dependent as well as time-dependent bactericidal activity. Initially bacterial killing by RIF was low but increased with time. The killing rate of RIF was higher compared to that of CLR that showed extremely low killing rate. Whereas after 21 days of exposure RIF and CLR were similar with respect to their bacteriostatic activity, both agents differ regarding the rate of bactericidal activity. After 3 days of exposure the bactericidal activity of RIF was less compared to that of CLR, whereas after 21 days RIF appeared superior over CLR. The low killing rate as seen for CLR was also observed for EMB.
It can be concluded that the antimicrobial agents differ with respect to time-dependent bacterial killing capacity. After long-term exposure of 21 days the comparative bactericidal capacity is highest for MXF, followed by AMK, RIF, CLR, and EMB, respectively. After short-term exposure of 3 days the comparative bactericidal capacity is highest for AMK, followed by MXF, CLR, and RIF or EMB. The rate of bacterial killing may have more clinical significance than the degree of killing, as in vivo after administration of the agents exposure to clinically achievable concentrations lasts for only a limited period of time. In addition, a rapid decrease in mycobacterial load is needed for therapeutic efficacy and also may result in a reduced risk of development of drug resistance. In this respect AMK seems to be superior, as it was the only agent that was rapidly bactericidal at relatively low concentrations which are far below the achievable plasma concentrations. These are important parameters which may compensate in part for the low intracellular penetrating capability of this agent.
RIF and MXF are bactericidal at concentrations below the concentrations achievable in plasma and tissues and inside infected cells. CLR inhibited bacterial growth at plasma concentrations attainable in vivo. Bacterial killing was only achieved at relatively high concentrations, and the killing rate of CLR is extremely low. As CLR concentrates intracellularly in macrophages and achieves excellent tissue penetration, it is bactericidal in mice and humans. Compared to CLR, higher concentrations of EMB were needed to obtain a bacteriostatic or bactericidal effect, which are far above the concentrations achievable in vivo. The relatively high concentration of EMB needed to obtain growth inhibition is within the range of MICs of EMB for most MAC isolates (20 to 32 mg/liter). EMB is considered bacteriostatic, and its principal role has been as a companion drug to prevent macrolide resistance.
Compared to the other aminoglycosides AMK appeared superior over STR, TOB, and GEN, which were active at only relatively high concentrations. This finding is in agreement with the general observation that AMK is the most active aminoglycoside against the nontuberculous mycobacteria (20
). Regarding the quinolones, MXF, particularly during the first 3 days of exposure, appeared superior over SPX and CIP. After 21 days of exposure, SPX and CIP showed similar bactericidal activity, but in terms of killing rate SPX is superior over CIP. The other agents investigated, INH, PZA, and AMC, were far less active. PZA was not bactericidal at all at the concentrations tested, whereas INH and AMC were bactericidal only at high concentrations that are not clinically relevant. It is generally known that these agents are not useful for treatment of MAC infections.
Based on the results of bacteriostatic capacity in terms of growth inhibition, CLR, RIF, AMK, and MXF all show substantial activity. According to the thresholds for interpretation of growth inhibition (23
), MAC strain 101 is considered “susceptible” to these agents. However, the present study demonstrates that the assay of time-dependent bactericidal capacity is more discriminative. The data show that these agents differ substantially with respect to their bactericidal capacity, particularly the killing rate, demonstrating a superior and rapid killing activity of AMK and high killing activity of MXF.
In the treatment of MAC, combinations of antimycobacterial agents are necessary to reduce the incidence of drug resistance. It is not well understood whether their use also results in improved efficacy in terms of enhanced eradication of mycobacteria. In the present study it was investigated whether addition of a number of antimycobacterial agents to CLR resulted in an increased level of bactericidal activity after 3 or 10 days of exposure in vitro against early-log-phase MAC. Significant enhancement of activity of CLR by combination with EMB could not be demonstrated. Only an additive effect has been shown. The addition of RIF or MXF as a third agent to the CLR-EMB combination did not significantly promote enhanced bacterial killing. Although the three-drug combinations resulted in higher bactericidal effect than any of the exposures used alone, these combinations did never reach a 1 log further decrease in mycobacterial numbers. Only AMK had a substantial effect by improving the activity of the combination CLR-EMB tenfold within only 3 days of exposure. This is in agreement with the findings of Fattorini et al., demonstrating that the addition of AK made the combination of CLR-EMB synergistic against a number of MAC strains in vitro (17
Other studies investigating the in vitro activity of CLR in combination with various antimycobacterial agents against MAC show discrepancy in results. Besides claims by some authors about a synergistic interaction between CLR in combination with EMB (20
), RIF (49
), EMB and RIF (41
), AMK (38
), or various quinolones (38
), only additive effects have been observed (20
). In addition, antagonism has been reported between CLR in combination with AMK (17
) or quinolones (49
). As the studies on the in vitro activities of CLR in two-drug combinations or three-drug combinations against MAC are controversial, these studies do not clearly provide insight into the rational design of combinations of agents with potent therapeutic activity.
Beta-lactam activity against mycobacteria has been described. However, the doses required to obtain efficacy in infection are not feasible. Synergistic activity of aminoglycoside-beta-lactam combinations towards bacterial strains is well known but was not investigated for mycobacteria. In the present study the addition of the beta-lactamase-stable AMC to AMK resulted in a higher level of MAC killing. In view of the low intracellular penetrating capability of aminoglycosides and beta-lactams, the clinical relevance of this finding with respect to intracellular MAC is questionable. However, as a result of their high antimicrobial activity, an aminoglycoside alone or in combination with a beta-lactam may be efficacious in eliminating MAC organisms which are growing extracellularly in the cavitating lesions in advanced stages of MAC infection.
There are many factors influencing the activity of antibiotics in vivo. Besides the antibiotic susceptibility of the infectious agent, the concentration profile of the antibiotic in serum, other body fluids, and tissues, and at the site of infection in relation to the dosing regimen is of importance. With respect to mycobacterial infections, antimicrobial activity against mycobacteria at the low growth rate is also an important determinant for therapeutic efficacy in view of the low metabolic activity of mycobacteria residing in tissues in the dormant state. The data from the present study show that, towards MAC that are not actively growing, only the quinolones exhibited bactericidal activity, although the absolute killing of these was substantially less compared to that of actively growing MAC. Even AMK, which was highly bactericidal against actively growing MAC, appeared not to be effective against MAC at the low growth rate. Whether this superior activity of quinolones for dormant Mycobacterium avium implies that quinolones should be used in the first line regimen for Mycobacterium avium disease will have to be clinically evaluated.