Mycobacterium avium Complex in Nude and Beige Mice ... value of the nude mouse model for the efficacy of chemotherapy is less than that of the beige mouse ...
ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, May 1997, p. 1168–1169 0066-4804/97/$04.0010 Copyright © 1997, American Society for Microbiology
Vol. 41, No. 5
Comparative Activities of Amikacin against Mycobacterium avium Complex in Nude and Beige Mice NACER LOUNIS, BAOHONG JI, CHANTAL TRUFFOT-PERNOT,
AND
JACQUES GROSSET*
Bacte´riologie et Virologie, Faculte´ de Me´decine, Pitie´-Salpeˆtrie`re, Paris, France Received 15 October 1996/Returned for modification 30 December 1996/Accepted 28 February 1997
After 4 weeks of treatment, clarithromycin (CLAR) and amikacin showed similar antimicrobial activities against the Mycobacterium avium complex in mice. There was a difference, however, in the effectiveness of the drugs in different types of mice: both drugs displayed bactericidal effects in beige mice but only bacteriostatic effects in nude mice. Because the effectiveness of CLAR is less in nude mice than in beige mice, the predictive value of the nude mouse model for the efficacy of chemotherapy is less than that of the beige mouse model. times weekly for 4 weeks; AMIK was administered by subcutaneous injection at a dosage of 100 mg/kg of body weight, and CLAR was given by esophageal cannula (gavage) at a dosage of 200 mg/kg. To monitor the multiplication of MAC, three to ten mice from untreated control groups were sacrificed on D21, D28, D35, and D45, whereas in the treated groups, the only sacrifice was carried out at the end of the 4-week treatment. To avoid any possible carryover of drugs in the organ suspensions (9), all surviving mice were sacrificed at D45, i.e., 72 h after administration of the last dose. To assess the severity of infection and the effectiveness of treatment, mortality rates, spleen weights, and numbers of CFU in the spleens and lungs were compared between the groups (9, 11). The results were analyzed by the Student t test and Fisher’s exact probability calculation. Differences were considered significant at the 95% level of confidence. A bacteriostatic effect was defined as a significant decrease in the mean number of CFU for the treated mice compared with that for the untreated control mice sacrificed concomitantly but no significant difference from the mean pretreatment value, i.e., the mean value obtained from untreated mice sacrificed at D14 after infection. A bactericidal effect was defined as a significant decrease in the mean number of CFU for the treated group from the pretreatment value. No mortality was observed for either mouse group before D14. During the period between D14 and D45, 3 (14.3%) of the 21 untreated beige mice died but none of the treated beige mice died, whereas for nude mice, except for a mouse treated with CLAR that was killed accidently during gavage, there was no mortality in either the control or the treated group. Therefore, at least during the initial 6 weeks after infection, the mortality rate of MAC-infected nude mice was very low and did not differ significantly from that of infected beige mice. As shown in Table 1, at D1 after infection the mean spleen weights were virtually the same, about 100 mg for beige and nude mice. Between D1 and D14, the spleen weights increased far more significantly in beige than in nude mice; by D14 the mean spleen weight in beige mice was six times greater than that at D1 and three times greater than that of nude mice (P , 0.01 for both differences). Thereafter, the spleen weights continued to increase for untreated mice but the increase was slower than that during the initial 2 weeks, and by D45 the mean spleen weight of beige mice was still three times greater than that of nude mice (P , 0.01). In brief, the splenomegaly was far more serious in MAC-infected beige mice than in infected nude mice. At the end of 4 weeks of treatment, the mean spleen weights of all treated groups of both mouse types
Disseminated infection caused by the Mycobacterium avium complex (MAC) is the most common systemic bacterial infection in patients with AIDS (10), occurring predominantly in those whose CD41 lymphocyte count is low. Using the beige mouse model, several investigators have clearly demonstrated that amikacin (AMIK) alone or in combinations displayed bactericidal activity against various strains of MAC (3, 6, 7, 9). Depending upon the duration of treatment (9), its activity was either slightly weaker than or similar to that of clarithromycin (CLAR), currently the key drug for the treatment of disseminated MAC infection in AIDS patients (1, 4, 5, 12). In addition, AMIK is clearly the only drug which is able to prevent the selection of MAC mutants resistant to CLAR in beige mice (9, 11). Nevertheless, the potential role of AMIK in the control of MAC infection and the prevention of the emergence of CLAR resistance in AIDS patients remains to be evaluated in a controlled clinical trial. Like all aminoglycosides, AMIK possesses two major disadvantages: parenteral administration and potential toxicity, especially ototoxicity and nephrotoxicity; consequently, before planning such a clinical trial, it is important to verify the promising effects of AMIK in other experimental models. Because the congenital athymic nude mouse (2) is deprived of activated T cells, we thought that its immune deficiency may be closer to that of patients with advanced HIV infection than the beige mouse (13), which is mainly deprived of NK cells. The objective of the present experiment was to determine the activities of AMIK and CLAR (as a positive control drug) against MAC in nude mice and to compare them with those in beige mice. Sixty female beige (C57B1/6J bgi/bgi) and 57 female outbred (Swiss) nude mice, 18 to 20 g of body weight, were each infected intravenously with 0.5 ml of a bacterial suspension containing 2.66 3 107 CFU of MAC strain 101. The MICs, as determined on 10% oleic acid–albumin–dextrose–catalase-enriched 7H11 agar, of AMIK and CLAR for strain 101 were identical, 16 mg/ml. On day 1 (D1) and D14 after infection, 5 and 10 infected mice, respectively, from both groups were sacrificed for enumeration of CFU in the spleens and lungs. At D14, the remaining mice were allocated randomly to an untreated control group of 21 beige or nude mice and two groups, with 12 beige or 10 to 12 nude mice per group, treated with AMIK or CLAR, respectively, alone. Drugs were given six
* Corresponding author. Mailing address: Bacte´riologie et Virologie, Faculte´ de Me´decine, Pitie´-Salpeˆtrie`re, 91 Blvd. de l’Ho ˆpital, 75634 Paris Cedex 13. Phone: 40-77-97-46. Fax: 45-82-75-77. 1168
VOL. 41, 1997
NOTES
TABLE 1. Mean spleen weights and organ CFU for beige and nude mice Groupa
n
Spleen weight (mg) 6 SE
CFU (log10) 6 SE in: Spleen
Lung
Beige mice Untreated control at D1 5 Untreated control at D14 10 Untreated control at D21 5 Untreated control at D28 5 Untreated control at D35 3 Untreated control at D45 5 CLAR treated at D45 12 AMIK treated at D45 12
101 6 6 622 6 25 674 6 156 736 6 63 943 6 109 870 6 129 395 6 21 419 6 27
6.07 6 0.13 7.91 6 0.05 8.15 6 0.12 8.42 6 0.08 9.10 6 0.18 9.07 6 0.17 6.89 6 0.07 7.03 6 0.04
4.36 6 0.04 6.09 6 0.09 6.38 6 0.26 7.03 6 0.14 7.63 6 0.37 7.66 6 0.13 4.94 6 0.09 5.41 6 0.11
Nude mice Untreated control at D1 5 Untreated control at D14 10 Untreated control at D21 4 Untreated control at D28 4 Untreated control at D35 3 Untreated control at D45 10 CLAR treated at D45 9 AMIK treated at D45 12
106 6 3 171 6 19 165 6 34 197 6 10 232 6 48 295 6 26 138 6 13 124 6 7
5.72 6 0.20 6.97 6 0.33 7.32 6 0.35 7.93 6 0.20 7.73 6 0.50 8.74 6 0.11 6.85 6 0.28 6.52 6 0.20
3.90 6 0.12 4.53 6 0.27 5.32 6 0.21 6.07 6 0.05 5.20 6 0.57 7.12 6 0.35 4.02 6 0.12 3.97 6 0.17
1169
These results confirmed that for nude mice AMIK displayed activity against MAC similar to that of CLAR. However, in contrast to the bactericidal activities demonstrated for the beige mice, both drugs displayed only bacteriostatic activities for the nude mice. Because the bactericidal activity of CLAR has been proven for AIDS patients (1, 4, 5, 12), the results indicate that CLAR and AMIK are less effective in the nude mouse model than in the beige mouse model and probably are also less effective in the nude mouse model than in AIDS patients. The discrepancy of the results between nude and beige mice might be attributed to the difference in their susceptibilities to MAC, as the CFU counts for beige mice were always greater than the corresponding values for nude mice. It seems our original hypothesis that the nude mouse is closer immunologically to AIDS patients than the beige mouse is oversimplified. As far as an animal model for testing the antiMAC activity of drugs is concerned, in spite of lacking functional T cells, the nude mouse model has less predictive value for the efficacy of chemotherapy (at least for CLAR and also possibly for AMIK) than the beige mouse model. This investigation was partially financed by Bristol-Myers Squibb, Paris, France.
a For treated groups, treatment began on D14 and the last dose was given on D42.
REFERENCES
were significantly smaller than the pretreatment values at D14 (P , 0.01) (Table 1). In terms of the ratios between the mean spleen weights of treated mice and the pretreatment values, the decrease of spleen weights did not differ significantly between mice treated with AMIK and with CLAR or between beige and nude mice, indicating that AMIK displayed an effect similar to that of CLAR in reducing the splenomegaly caused by MAC infection in either mouse type. On D1 after infection, MAC bacteria were isolated from spleens and lungs of all sacrificed beige and nude mice. In untreated control mice, the mean number of CFU in both organs of both mouse types progressively increased (Table 1) and the increases during each fortnight (i.e., between D1, D14, D28, and D45) almost invariably attained statistical significance (P , 0.05 or , 0.01); in agreement with the results of our earlier studies (8), the CFU counts for beige mice were always greater than the corresponding values for nude mice, although the differences did not always attain statistical significance. Compared with the values for beige mice, the individual variations of the CFU counts were much greater for nude mice (Table 1), most likely because the nude mice were outbred, whereas the beige mice were inbred. As observed in previous experiments (9, 11), the mean numbers of CFU in the spleens were significantly greater than those in the lungs (P , 0.01) for both mouse types. On D45, i.e., after a 4-week treatment, except for the spleens of mice treated by CLAR, the mean numbers of CFU per organ in beige mice were always greater than in nude mice treated with the same drug, a finding consistent with the greater CFU counts in untreated beige mice than in untreated nude mice. Except for the lungs of beige mice, the CFU counts per organ among mice treated by AMIK did not differ significantly from those among mice treated by CLAR and were 2 to 3 log10 units smaller than those of untreated controls sacrificed concomitantly (Table 1), indicating that both drugs were similarly active in mice of both types. The mean numbers of CFU in the treated groups were also smaller than the corresponding pretreatment values at D14; nevertheless, the differences reach statistical significance only for beige mice (P , 0.01), not for nude mice.
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