Susceptibilities of Mycobacterium malmoense determined at the growth optimum pH (pH 6.0).

SETTING: Pulmonary disease caused by Mycobacterium malmoense is increasing. Conventional in vitro antimicrobial susceptibilities correlate poorly with response to treatment for this organism. Radiometrically determined minimum inhibitory concentrations (MICs) allow quantitative susceptibility testing for non-tuberculous mycobacteria. The M. avium complex (MAC) has been investigated extensively with this approach, and clear interpretative criteria have been established at pH 6.8. However, there has been little work with the acidophilic M. malmoense, which grows poorly at pH 6.8. OBJECTIVE: To determine whether MICs at pH 6.0 provide results compatible with the interpretative criteria established for the MAC. DESIGN: MICs were performed in Middlebrook PZA medium (pH 6.0) and 7H12 medium (pH 6.8) for ten strains of M. malmoense. RESULTS: MICs can be determined at pH 6.0 for M. malmoense using the criteria adopted for the M. avium complex. CONCLUSION: The low optimal pH of M. malmoense suits this organism for growth in acid conditions. As with MAC, M. malmoense multiplies within macrophages in vivo, and MICs determined at pH 6.0 may reflect in vivo activity. The combination of radiometric MIC testing at optimal growth pH and interpretation based on pharmacokinetic parameters may be helpful in designing therapeutic regimens.

Rapid broth macrodilution method for determination of MICs for Mycobacterium avium isolates.

A multicenter study was done to investigate the accuracy and reproducibility of a method for determining the MICs of antimicrobial agents against the Mycobacterium avium complex in 7H12 broth with the BACTEC system. In phase I, with eight drugs and 10 strains, intralaboratory reproducibility was 95.7 to 100%, allowing a 1-dilution difference upon repeat testing. The results of phase II testing with 41 additional strains were consistent with those obtained in phase I, with good interlaboratory reproducibility. The radiometric method was validated by sampling and plating of the same broth cultures and determining, by the number of CFU per milliliter, the lowest drug concentration that inhibited more than 99% of the initial bacterial population. Three test concentrations of each drug and the tentative interpretation of results are proposed. Radiometric MIC determination has the potential to become the method of choice for clinical microbiology laboratories and evaluation of new agents for the treatment of M. avium infections, both pulmonary and disseminated.

Bacteriostatic and bactericidal activities of gentamicin alone and in combination with clarithromycin against Mycobacterium avium.

The inhibitory activity of gentamicin against Mycobacterium avium depended on the pH of the medium, and the broth-determined MICs for 90% of strains were 5.0 micrograms/ml at pH 7.4, 9.5 micrograms/ml at pH 6.8, and greater than 16.0 micrograms/ml at pH 5.0. The MBCs were two- to eightfold higher than the MICs. The combined effect of gentamicin and clarithromycin was additive, and the MICs and MBCs of each drug were either the same as those in the single-drug tests or reduced twofold.

Clarithromycin minimal inhibitory and bactericidal concentrations against Mycobacterium avium.

Minimal inhibitory and bactericidal concentrations (MIC and MBC) of clarithromycin were determined with 49 Mycobacterium avium strains isolated from patients with acquired immunodeficiency syndrome. The inhibitory activity depended on the pH of the medium: the drug was more active at pH 7.4 and less active at pH 5.0, with activity at pH 6.8 in an intermediate position. The broth-determined MIC found at pH 7.4 were 0.25 and 0.5 micrograms/ml for most strains. The agar-determined MIC for most strains ranged from 1.0 to 4.0 micrograms/ml. The MBC of the drug were 8- to 64-fold higher than the MIC, which indicates that the efficacy of clarithromycin can be associated with its inhibitory rather than its bactericidal activity.

Comparison of bacteriostatic and bactericidal activity of isoniazid and ethionamide against Mycobacterium avium and Mycobacterium tuberculosis.

Minimal inhibitory and bactericidal concentrations (MIC and MBC) of isoniazid and ethionamide were determined in 7H12 broth in experiments with 68 Mycobacterium avium strains and 14 wild drug-susceptible M. tuberculosis strains. MICs of isoniazid for M. tuberculosis were from 0.025 to 0.05 microgram/ml, and for M. avium from 0.6 to greater than 10.0 micrograms/ml. MICs of ethionamide for M. tuberculosis were from 0.3 to 1.25 micrograms/ml, and 42.7% of M. avium strains were within the same range. Isoniazid and ethionamide were highly bactericidal against M. tuberculosis, but they had very low bactericidal activity against M. avium.

Thiacetazone: in vitro activity against Mycobacterium avium and M. tuberculosis.

Bacteriostatic and bactericidal activity of thiacetazone was determined for 68 M. avium clinical isolates and 14 wild drug-susceptible M. tuberculosis strains. The drug had equally low bactericidal activity against both mycobacterial species. The inhibitory activity against most of the M. avium strains was greater than it was against M. tuberculosis. The broth determined MICs for 65 of 68 M. avium strains were between 0.02 and 0.15 micrograms/ml, while the MICs for M. tuberculosis ranged from 0.08 to 1.2 micrograms/ml.

MICs and MBCs of Win 57273 against Mycobacterium avium and M. tuberculosis.

A new quinolone, Win 57273 [1-cyclopropyl-7-(2,6-dimethyl-4-pyridinyl)-6-fluoro-1,4-dihydro-4-oxo-3 - quinolonecarboxylic acid], synthesized by Sterling Research Group, was tested in vitro against Mycobacterium tuberculosis and Mycobacterium avium strains. The broth-determined MICs of this agent ranged from 1.0 to 4.0 micrograms/ml for M. tuberculosis strains and from 0.25 to 8.0 micrograms/ml for M. avium strains. A distinctive feature of this agent, in comparison with ofloxacin and ciprofloxacin, is its substantially greater activity at the low pHs. For M. avium strains, the MICs of Win 57273 were 2.0 micrograms/ml or less for 54.5% of strains at pH 6.8 and 85.5% of strains at pH 5.0. Win 57273 was more active than ciprofloxacin against M. avium strains, and this difference was very substantial for all M. avium strains at pH 5.0. Taking into account that the predominant locations of these organisms in vivo are within the phagosomes and phagolysosomes of macrophages, i.e., in acidic environments at pH 5.0 or lower, the greater activity of Win 57273 at low pH makes this quinolone especially promising for M. avium infection. The bactericidal activity of Win 57273 for M. avium strains was the same as that of ciprofloxacin, with MBCs from 4.0 to 16.0 micrograms/ml.

Bactericidal activity in vitro of various rifamycins against Mycobacterium avium and Mycobacterium tuberculosis.

Minimal inhibitory and bactericidal concentrations (MICs and MBCs) of rifampin (RMP), rifabutin (RBT), rifapentine (RPT), CGP-7040, and P-DEA, were determined for 50 M. avium strains in 7H12 liquid medium radiometrically under various pH conditions. Half were isolated from patients with AIDS and the other half from patients without AIDS but with pulmonary disease. The MICs and MBCs were also determined in 7H12 broth for M. tuberculosis strains. The MIC results obtained with M. tuberculosis strains, and the serum peak levels in humans, were used as standards for interpretation of the MICs and MBCs of the rifamycins for M. avium. The bactericidal activity of all rifamycins for M. avium was substantially lower than for M. tuberculosis. The majority of M. avium strains was within the "susceptible" category, e.g., comparable to susceptible M. tuberculosis strains, when tested with CGP-7040 and RPT, and all of them were "moderately susceptible" when tested with P-DEA. On the basis of in vitro bacteriostatic and bactericidal activity, it seems that three agents, RPT, P-DEA, and CGP-7040 have more potential than do RMP and RBT against M. avium disease.

Is pyrazinamide bactericidal against Mycobacterium tuberculosis?

Bactericidal activity of pyrazinamide (PZA) was tested at pH 5.6 in 7H12 broth against drug-susceptible M. tuberculosis strains. The highest tested concentrations of PZA, 500 and 1,000 micrograms/ml, killed no more than 76% of the bacterial population. These concentrations are more than 32 times greater than the minimal inhibitory concentration (MIC) and the achievable in vivo concentrations. Despite high clinical efficacy of PZA and its so-called sterilizing activity in mouse experiments, this drug is much less bactericidal in vitro than any other known antituberculosis drug.

Minimal inhibitory concentrations of rifabutin, ciprofloxacin, and ofloxacin against Mycobacterium tuberculosis isolated before treatment of patients in Taiwan.

Minimal inhibitory concentrations (MICs) of rifabutin, ciprofloxacin, and ofloxacin were determined for "wild" Mycobacterium tuberculosis strains, susceptible to all antituberculosis drugs in the conventional test, isolated from newly diagnosed Taiwanese patients who had never had prior treatment for tuberculosis. These MICs were within the limits previously reported for strains isolated in the United States. The range of MICs of rifabutin for Taiwanese strains was 0.015 to 0.125 micrograms/ml; ciprofloxacin, 0.25 to 2.0 micrograms/ml; ofloxacin, 0.5 to 2.0 micrograms/ml. On the basis of an evaluation of the highest broth-determined MICs found in this and in a previous study, we suggest that the following MICs, when determined in 7H12 broth radiometrically, should be used as breakpoints to classify the strain as "susceptible": for rifabutin, 0.125 micrograms/ml or less; for ofloxacin and ciprofloxacin, 2.0 micrograms/ml or less.

Does pyrazinoic acid as an active moiety of pyrazinamide have specific activity against Mycobacterium tuberculosis?

The commonly accepted hypothesis explaining the mechanism of action of pyrazinamide (PZA) is based on the assumption that PZA-susceptible Mycobacterium tuberculosis strains produce pyrazinamidase, which hydrolyzes PZA to the antibacterial moiety pyrazinoic acid (POA). It is not clear whether POA has specific antimicrobial activity or the inhibition of growth caused by POA is due to its ability to lower the pH of the environment below the limits of tolerance of M. tuberculosis growth. We confirmed in this study that POA, depending on the concentration, lowered the pH of 7H12 broth (pH 6.0), which ranged from 5.8 at 120.0 micrograms/ml to 4.6 at 960.0 micrograms/ml. Therefore, we tested the inhibitory effects of different concentrations of POA in broth in which the final pH was adjusted to 5.6 by adding appropriate amounts of phosphoric acid or dipotassium phosphate. Under these conditions, we found a clear dose-response correlation, proving that POA does have specific antimicrobial activity. The MIC of POA at pH 5.6 was 240 to 480 micrograms/ml, 8- to 16-fold higher than the MIC of PZA under the same conditions and much higher than the concentrations achievable in humans. This suggests that the action of POA in an acid environment is a combined effect of its specific activity and its ability to lower the pH below the limits of tolerance of the target organism.

Clofazimine and other rimino-compounds: minimal inhibitory and minimal bactericidal concentrations at different pHs for Mycobacterium avium complex.

Clofazimine (Lamprene, B663) and 11 rimino-compounds were tested for activity against Mycobacterium avium by finding the minimal inhibitory concentrations (MIC) of these drugs in liquid medium. The activity of these compounds was affected in acidic conditions (pH 6.0 and 5.0), but the MICs observed even at pH 5.0 were significantly lower than the concentrations achievable in macrophages. This information is especially useful in the light of four important facts: that these drugs tend to accumulate within macrophages; that M. avium tends to multiply within macrophages; that this intracellular environment has a low pH; and that M. avium tolerates these low pH conditions. Such data confirm the expectation that rimino-compounds inhibit the intracellular M. avium bacterial population. The minimal bactericidal concentrations (MBC) of clofazimine and B746, the most active analogue, were from 64 to 256 times higher than the MIC, but it is not clear whether these drugs can accumulate within the macrophages in concentrations high enough to produce the bactericidal effect as well.

Combinations of rifampin or rifabutine plus ethambutol against Mycobacterium avium complex. Bactericidal synergistic, and bacteriostatic additive or synergistic effects.

Ethambutol, when combined with rifampin or rifabutine (ansamycin, LM427), produced an additive inhibitory effect against most of the tested M avium complex strains (12 of 16 with rifampin, 13 of 16 with rifabutine). The inhibitory effect was synergistic for the remaining 4 of 16 and 3 of 16 strains. This relationship reduced the minimal inhibitory concentrations for each drug in combination. In addition, the minimal bactericidal concentrations of all 3 drugs were reduced because of synergistic bactericidal activity in 6 of 6 strains tested.

Rifabutine: minimal inhibitory and bactericidal concentrations for Mycobacterium tuberculosis.

The minimal inhibitory concentrations (MIC) and minimal bactericidal concentrations (MBC) of rifabutine (ansamycin LM427, spiropiperidyl rifamycin) against rifampin-susceptible and rifampin-resistant strains of Mycobacterium tuberculosis were determined. The MICs for 17 rifampin-susceptible strains were low, falling in a narrow range of 0.06 microgram/ml or less when determined by either the agar-dilution method (in 7H10 or 7H11 agar) or in 7H12 broth (radiometrically or by sampling and colony-forming units [CFU/ml] determination). The MICs of rifabutine for 21 rifampin-resistant strains were much higher, ranging from 0.25 to 16.0 micrograms/ml. The MBCs of rifabutine were 0.125 or 0.25 microgram/ml for rifampin-susceptible strains, and were 4.0 and 32.0 micrograms/ml for two tested rifampin-resistant strains. The MIC/MBC ratio was 1:4 for rifampin-susceptible strains.

Bacteriostatic and bactericidal activity of ciprofloxacin and ofloxacin against Mycobacterium tuberculosis and Mycobacterium avium complex.

MICs of ciprofloxacin were lower than MICs of ofloxacin for both M. tuberculosis and M. avium complex strains. The MICs of both drugs for 41 M. tuberculosis strains were in a narrow range, and significantly lower than the achievable serum concentrations. The MICs of ciprofloxacin for 46 M. avium strains were in a wide range, and in only 28% were the broth-determined MICs of ciprofloxacin within the same range as for M. tuberculosis and lower than the achievable serum level. When compared with the concentrations achievable in macrophages, the broth-determined MICs 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 MIC/MBC ratio.

Determination of MICs of conventional and experimental drugs in liquid medium by the radiometric method against Mycobacterium avium complex.

The aim of this study was to search for new drugs active against the Mycobacterium avium complex and to re-examine the activity of some conventional antituberculosis drugs against these species. This progress report describes the protocol and phases of in vitro experiments in a search which included 57 different compounds tested against numerous strains of M. avium clinical isolates. The preliminary screening and MIC determination of these drugs were conducted in 7H12 broth by the radiometric method (BACTEC system). Of the total of 57 drugs, 23 were discarded after preliminary screening and 17 after MIC determination. The remaining 17 drugs were considered sufficiently promising for more detailed in vitro studies. These, now in progress, include MIC determination by two additional methods (in broth by sampling and plating and in 7H11 agar plates), MBC determination and drug combination studies. The following drugs are currently undergoing these detailed studies: isoniazid, rifampin, rifabutine (ansamycin LM427), amikacin, streptomycin, ethambutol, ethionamide, cycloserine, clofazimine (CF), CF derivative B746, CF derivative B1865, ofloxacin, ciprofloxacin, cephalosporin BMY 28142, trimethoprim-sulfamethoxazole, spectinomycin derivative U6633F(B), and dihydromycoplanecin.

Pyrazinamide is not active in vitro against Mycobacterium avium complex.

Strains of M. tuberculosis that elaborate pyrazinamidase are typically susceptible in vitro and in vivo to pyrazinamide (PZA). However, we found that 33 strains of M. avium complex (MAC), all of which were pyrazinamidase-positive, were resistant in vitro to a high concentration (100 micrograms/ml) of PZA when tested at low pH in 7H12 broth by radiometric (BACTEC) method. The drug was equally ineffective against these bacteria within cultured normal human macrophages. We conclude that the pyrazinamidase test is not suitable for susceptibility studies against M. avium complex. On the basis of our in vitro studies, which support earlier data, we believe that PZA is not appropriate for therapy of MAC disease.

Ethambutol MICs and MBCs for Mycobacterium avium complex and Mycobacterium tuberculosis.

We determined the MICs of ethambutol for both Mycobacterium avium and Mycobacterium tuberculosis strains by using broth dilution (7H12 broth, radiometric method) and agar dilution (7H11 agar) methods. We found the MICs to be much lower in liquid than in solid medium. The broth-determined MICs for susceptible M. tuberculosis and most of the M. avium strains were comparable to the levels in blood of patients, being lower than the peak levels. We propose that the MICs, determined radiometrically in in 7H12 broth, be considered as tentative criteria for susceptibility testing of M. avium isolates in future clinical trials. The use of these values instead of critical concentrations should also be considered as an alternative to the conventional susceptibility testing method in chemotherapy of tuberculosis. Ethambutol produced bactericidal effects against both M. tuberculosis and M. avium, and the MIC/MBC ratios were in the same range for both species when MICs and MBCs were tested in 7H12 broth by conventional sampling and plating.

Determination of ansamycin MICs for Mycobacterium avium complex in liquid medium by radiometric and conventional methods.

A radiometric method to determine the MIC of ansamycin (LM427) for Mycobacterium avium complex clinical isolates has been developed. It is based on a comparison of the conventional growth curve determination and the radiometric detection of growth (growth index) in the same liquid medium (7H12 broth). This new method requires less time and labor than does a conventional determination of MIC in liquid medium (CFU). Other advantages of this method include relatively short periods of exposure of the drug to 37 degrees C and the composition of 7H12 broth, which has practically no substrates which could absorb or bind the drug. Thus, a more accurate estimation of the MIC in this medium can be expected than by the conventional agar dilution (proportion) method. The MICs of ansamycin appeared to be higher in agar plates than in 7H12 broth. More than 70% of the isolates had a broth-determined MIC one to three times lower than the average peak concentration of ansamycin achieved in sera of patients. The wide range of MICs suggests the importance of testing susceptibility in broth with many concentrations in addition to, or rather than in, agar plates with concentrations of 2.0 or 1.0 micrograms/ml only. Taking into account relatively low levels of ansamycin in sera of patients, it would be appropriate to compare the MICs with the levels in serum to make the outcome of chemotherapy more predictable.

Determination of in vitro susceptibility of Mycobacterium tuberculosis to cephalosporins by radiometric and conventional methods.

Among eight cephalosporins and cephamycins tested in preliminary in vitro screening against Mycobacterium tuberculosis, the most promising for further study was found to be ceforanide, followed by ceftizoxime, cephapirin, and cefotaxime. Moxalactam, cefoxitin, cefamandole, and cephalothin were found to be not active enough against M. tuberculosis to be considered for further in vitro studies. The antibacterial activity of various ceforanide concentrations was investigated by three methods: (i) the dynamics of radiometric readings (growth index) in 7H12 broth; (ii) the number of CFU in the same medium; and (iii) the proportion method on 7H11 agar plates. There was a good correlation among the results obtained with these methods. The MIC for most strains ranged from 6.0 to 25.0 micrograms/ml. The BACTEC radiometric method is a reliable, rapid, and convenient method for preliminary screening and determination of the level of antibacterial activity of drugs not commonly used against M. tuberculosis.