Carryover of clofazimine into culture media

While assessing the chemotherapeutic potential of various agents againstMycobacterium avium complex (MAC) infection in beige mice, Ji et al. (6) expressed concern about the possible carryover of clofazimine (CFM) into the culture media used for enumerating CFUs. They stated that the presence of the carried-over drug inhibits the growth of the organisms present in the tissue homogenates, thus giving a false impression of in vivo activity. Since we are also actively involved in studies with this drug for its anti-MAC and antituberculosis activities (1–5) we had similar questions about this drug’s presence in the culture media creating a potential source of an artifact of results. We have therefore carried out four different assessments to answer this question. First we measured the concentrations of the drugs in liver and lungs during various periods of treatment and calculated the amounts that could be transferred to the media during the process of enumeration of the CFUs (5). The highest concentration that could be seen in our studies (5) was 99.7 mg/g of tissue, which amounts to 149.5 mg in the whole liver, which is homogenized in 10.0 ml of saline. A 1/10 dilution of the wholeliver homogenate in 10.0 ml of saline, when transferred to a 25-ml 7H11 plate, results in a concentration of 0.0059 mg/ml on the plate, which is far below the MIC of the drug for MAC or M. tuberculosis. Similar calculations for the lung tissue showed only nanogram quantities that could be transferred to the plates. Ji et al. (6) have also presented data on the levels of CFM present in various organs at 12 weeks of treatment. In fact, their figures are considerably lower than what we obtained at various periods up to 61 days. However, it is difficult from their data to estimate the carried-over drug levels, since the volume of the Lowenstein-Jensen media used in their study was not given. Second, we could strongly infer the lack of carryover by looking at the CFU obtained from plates inoculated with various dilutions of the tissue homogenates. In all our studies, we use three 10-fold dilutions of the homogenates, and for calculations of the total CFU in the whole organ, we choose readings of 20 to 100 colonies in appropriate plates and use the necessary multiplication factors for the volume (0.1 ml) and dilution of the inoculum. Invariably, the CFU counts decreased in an approximately 10-fold fashion, parallel to the 10-fold dilutions of the suspensions spread on the 7H11 plates. If, on the other hand, there is carryover of the drug, one should get the lowest CFUs at the lowest dilution (i.e., 10), with a corresponding increase as the dilution progresses (e.g., to 10 or 10, etc.); such a situation was never encountered in our many studies with CFM. The argument of Ji et al. (6) that the tissue homogenates could not be appropriately diluted for want of a water-soluble organic solvent would not be tenable, since the CFUs with the various dilutions were not similar but were reduced in a stepwise fashion. It is also necessary to point out that using low numbers of 1 or 2 colonies for the calculation of CFUs in the whole organ, as has been used by Ji et al. (6), may not yield definitive information. Third, in some reconstitution experiments in which we added known numbers of organisms to homogenates of tissues from CFM-treated or control mice and enumerated the CFU by plating 0.1-ml aliquots of appropriate dilutions, we found no difference between the two sources of tissues. Had there been any carryover of the drug, the CFM-treated animals should have shown a definitive reduction of CFU counts. Finally, in some experimental chemotherapy studies against M. tuberculosis challenges, wherein mortality and CFU counts could be used as valuable parameters, we found no mortality in CFM-treated animals, while all (100%) of the untreated controls died within 4 weeks. The organ weights of the drugtreated animals were normal, though the organs were hyperpigmented (the usual appearance after treatment with this drug), while the organs from control animals were markedly enlarged. These observations were parallel to the gross reduction of CFU counts in the drug-treated group compared with that in the controls. The wealth of literature available on the role of CFM in the chemotherapy of MAC in humans should also vouch for the chemotherapeutic potential of this drug. On the basis of these observations, we are unable to share the concerns of Ji et al. (6) that the activity of CFM is overestimated because of a carryover phenomenon.