Received 6 October 1995/Returned for modification 27 November 1995/Accepted 29 January 1996

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1 ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Apr. 1996, p Vol. 40, No /96/$ Copyright 1996, American Society for Microbiology Activities of the Glycylcyclines N,N-Dimethylglycylamido-Minocycline and N,N-Dimethylglycylamido-6-Demethyl-6-Deoxytetracycline against and Tetracycline-Resistant Isolates of Rapidly Growing Mycobacteria BARBARA A. BROWN, 1 * RICHARD J. WALLACE, JR., 1,2 AND GRACE ONYI 1 Department of Microbiology 1 and Center for Pulmonary Infectious Disease Control, 2 University of Texas Health Center, Tyler, Texas Received 6 October 1995/Returned for modification 27 November 1995/Accepted 29 January 1996 Susceptibilities to the new semisynthetic tetracycline (Tet) compounds N,N-dimethylglycylamido-minocycline (DMG-MINO) and N,N-dimethylglycylamido-6-demethyl-6-deoxytetracycline (DMG-DMDOT) were compared with those to doxycycline, minocycline, and Tet for 198 Tet-resistant (Tet r ) and 33 Tet-susceptible (Tet s ) clinical isolates of rapidly growing mycobacteria (RGM) including the Mycobacterium fortuitum group, Mycobacterium abscessus, Mycobacterium chelonae, and Mycobacterium mucogenicum and 68 isolates belonging to six taxa of All Tet r RGM were highly susceptible to the glycylcyclines. The MICs at which 50 and 90% of isolates are inhibited were <0.125 and <0.25 g/ml, respectively, for DMG-DMDOT and <0.25 and 1 g/ml, respectively, for DMG-MINO. The MIC of DMG-DMDOT at which 50% of Tet r strains are inhibited was the same as that for Tet s strains for each of the four taxa of RGM. The new agents were less active against Nocardia spp. MICs of DMG-DMDOT were comparable to those of minocycline except for the MICs for Nocardia brasiliensis sensu stricto, the new taxon Nocardia pseudobrasiliensis, and some isolates of Nocardia nova, against which they were four- to eightfold more active. The MICs of DMG-DMDOT were consistently lower than those of DMG-MINO for RGM. This class of drugs offers exciting therapeutic potential for RGM and for selected species of Nocardia. * Corresponding author. Mailing address: Department of Microbiology, University of Texas Health Center at Tyler, P.O. Box 2003, Tyler, TX Phone: (903) Fax: (903) Nocardia and the rapidly growing mycobacteria (RGM) are environmental organisms that cause both cutaneous and pulmonary infections (1, 8, 10, 18, 26, 28 31, 33, 34). Because of the chronic nature of these diseases, 3 to 6 months of therapy is usually required. This dictates the need for effective oral antimicrobial agents (1, 8, 18, 26, 28, 33). Both groups of organisms are relatively resistant to drugs, including most oral -lactams and tetracyclines, with sulfonamides, the macrolides, and the newer quinolones providing the best potential for oral therapy. Not all species or even isolates within a species are susceptible to any one of these classes of drugs. Recent treatment of diseases caused by RGM has focused on the possible role of the new macrolides azithromycin and clarithromycin (6, 27, 33). Treatment of infections caused by, especially with oral agents, has evolved very little in the past several decades. Now, however, a new class of semisynthetic tetracyclines referred to generically as the glycylcyclines (25) is on the horizon. Two of these semisynthetic derivatives, one containing the N,N-dimethylglycylamido substituent at the 9 position of minocycline (DMG-MINO) and N,N-dimethylglycylamido- 6-demethyl-6-deoxytetracycline (DMG-DMDOT), are undergoing investigation. In the study described here we compared the in vitro activities of these two new glycylcyclines by the broth microdilution method with those of tetracycline, doxycycline, and minocycline against four types of RGM including the Mycobacterium fortuitum group, Mycobacterium abscessus, Mycobacterium chelonae, and Mycobacterium mucogenicum (formerly the Mycobacterium chelonae-like organism [21, 30]) and six different taxa of Nocardia including Nocardia nova, Nocardia brasiliensis sensu stricto, the new taxon within Nocardia brasiliensis, Nocardia pseudobrasiliensis (17, 26), Nocardia farcinica, Nocardia otitidiscaviarum, and the type six susceptibility group within the N. asteroides complex (22, 29, 31, 33). MATERIALS AND METHODS Isolates. One hundred ninety-eight clinical isolates of RGM which were tetracycline resistant (Tet r ) (defined as an MIC of 8 g/ml) for any of the tetracyclines) including isolates of the M. fortuitum group (45 strains), M. abscessus (91 strains), M. chelonae (49 strains), and M. mucogenicum (13 strains); 33 tetracycline-susceptible (Tet s ) strains of RGM of the same four groups; and 68 clinical isolates of, including 20 N. brasiliensis sensu stricto isolates (excluding the recently described strain N. pseudobrasiliensis) (17, 26), 12 N. farcinica isolates, 7 N. otitidiscaviarum isolates, 19 N. nova isolates, and 10 type 6 N. asteroides complex isolates submitted to the Mycobacteria/Nocardia Research Laboratory at the University of Texas Health Center at Tyler between 1994 and 1995 for susceptibility testing, were evaluated. Isolates of RGM and Nocardia were from respiratory specimens, skin and soft tissue sites, blood, and a small number of other miscellaneous sources. Seven reference strains of RGM and were obtained from the American Type Culture Collection (ATCC), Rockville, Md.; these included Mycobacterium peregrinum ATCC and ATCC 14467, M. abscessus ATCC 19977, N. nova ATCC 33726, Nocardia farcinica ATCC 3318, N. brasiliensis ATCC 19296, and N. otitidiscaviarum ATCC Five other reference strains, M. mucogenicum ATCC 49650, M. fortuitum unnamed third biovariant complex ATCC and ATCC 49404, and the new taxon N. pseudobrasiliensis ATCC and ATCC (17, 26), were taken from stocks stored at 70 C used for submission of these strains to ATCC. Isolates were identified as belonging to the M. fortuitum or M. chelonae- M. abscessus group by the referring laboratories by standard methods (19). Isolates were further classified by their antibiotic susceptibility patterns, including susceptibility to polymyxin B (32). The M. fortuitum group included isolates of M. fortuitum (previously M. fortuitum biovar fortuitum), M. fortuitum unnamed third biovariant, and M. peregrinum (previously M. fortuitum biovar peregrinum). M. chelonae and M. abscessus were differentiated by using differences in susceptibility to tobramycin and cefoxitin and the ability to utilize citrate as a sole 874

2 VOL. 40, 1996 GLYCYLCYCLINE ACTIVITIES 875 TABLE 1. MIC ranges and MIC 50 s and MIC 90 s of tetracycline, minocycline, doxycycline, and the new glycylcycline DMG-DMDOT for clinical Tet r RGM isolates Organism Antimicrobial agent a No. of isolates tested M. fortuitum group b TET MINO DOXY DMG-DMDOT M. chelonae TET MINO DOXY DMG-DMDOT M. abscessus TET MINO DOXY DMG-DMDOT M. mucogenicum TET MINO DOXY DMG-DMDOT b The M. fortuitum group includes M. fortuitum, M. peregrinum, and M. fortuitum unnamed third biovariant complex. carbon source (16, 23, 28, 33). Nocardia isolates were identified by standard biochemical testing (29, 31, 33) and susceptibility patterns (26, 29, 31, 33). Susceptibility testing. Initial susceptibility testing of the new compounds was done with DMG-MINO (Lederle Laboratories, Pearl River, N.Y.) by a previously described broth microdilution MIC method (3, 4, 25). Serial twofold dilutions of the antimicrobial agents including tetracycline, doxycycline, minocycline, and the new compound, DMG-MINO, were prepared as directed by the manufacturer in cation-supplemented Mueller-Hinton II broth (Becton-Dickinson Microbiology Systems, Cockeysville, Md.) to concentrations of 0.5 to 64 g/ml for tetracycline, 0.25 to 16 g/ml for doxycycline (Pfizer Laboratories, Groton, Conn.), 1 to 64 g/ml for minocycline (Lederle Laboratories), and 0.25 to 32 g/ml for DMG-MINO. Subsequently, clinical development of a second glycylcycline was begun so susceptibility testing was switched to DMG-DMDOT. Commercial custom-prepared plates (Sensititre; Radiometer America, West Lake, Ohio) with DMG- DMDOT (Lederle Laboratories) were used. Final concentrations of tetracycline at1to128 g/ml, doxycycline at 0.25 to 128 g/ml for RGM and 0.5 to 64 g/ml for, minocycline at 0.5 to 128 g/ml for RGM and 1 to 128 g/ml for, and DMG-DMDOT at 0.06 to 128 g/ml for RGM and 0.25 to 64 g/ml for were tested. Three sets of MIC plates were prepared; noncommercial plates with the DMG-MINO derivative were inoculated as described previously (3, 4, 22). The commercial custom-prepared plates were inoculated according to the manufacturer s directions. MICs were read after 72 h of incubation in room air (3, 4) at 30 C for RGM and 35 C for Because both DMG-DMDOT and DMG-MINO were not tested against most isolates, a direct comparison of the activities of the two compounds was not possible. The data are therefore presented in two separate tables. Quality control. Quality control was performed with Staphylococcus aureus ATCC 29213, Enterococcus faecalis ATCC 29212, Escherichia coli ATCC 25922, and Pseudomonas aeruginosa ATCC Suggested quality control ranges for MICs were those provided by each manufacturer and the National Committee for Clinical Laboratory Standards for tetracycline and minocycline (13). Quality controls for doxycycline MICs included S. aureus ATCC (MIC range, 0.25 to 1 g/ml) and E. coli ATCC (MIC range, 1 to 4 g/ml). Ranges for the glycylcyclines were proposed by using data supplied by the manufacturer. They were as follows: for S. aureus ATCC 29213, MIC range, to 0.5 g/ml; for E. faecalis ATCC 29212, MIC range, 0.03 to 0.25 g/ml; for E. coli ATCC 25922, MIC range, to 0.5 g/ml); and for P. aeruginosa ATCC 27853, MIC range, 4 to 16 g/ml. MICs for the bacterial strains were read after 24 h of incubation at 35 C. RESULTS A summary of MIC ranges and the MICs of tetracycline, doxycycline, minocycline, and the two new glycylcyclines at which 50% (MIC 50 s) and 90% (MIC 90 s) of the RGM and Nocardia isolates are inhibited is provided in Tables 1, 2, and 3. A summary of the MIC results for the reference strains of RGM and Nocardia is presented in Table 4. A total of 198 Tet r isolates of RGM were tested (for one isolate of M. fortuitum, the tetracycline MIC was 4 g/ml and the doxycycline MIC was 8 g/ml); for all other isolates, tetracycline MICs were 8 g/ml). Because three sets of MIC plates were used, the MICs of both DMG-MINO and DMG- TABLE 2. MIC ranges and MIC 50 s and MIC 90 s of tetracycline, minocycline, doxycycline, and the new glycylcycline DMG-MINO for clinical Tet r RGM isolates Organism Antimicrobial agent a No. of isolates tested b M. fortuitum group c TET MINO DOXY DMG-MINO M. chelonae TET MINO DOXY DMG-MINO M. abscessus TET MINO DOXY DMG-MINO M. mucogenicum TET MINO DOXY DMG-MINO b Note that MIC 50 s and MIC 90 s were not computed when the number of isolates tested was less than 10. c The M. fortuitum group includes M. fortuitum, M. peregrinum, and M. fortuitum unnamed third biovariant complex.

3 876 BROWN ET AL. ANTIMICROB. AGENTS CHEMOTHER. TABLE 3. MIC ranges and MIC 50 s and MIC 90 s of tetracycline, minocycline, doxycycline, and the new glycylcycline DMG-DMDOT for clinical Nocardia isolates Organism Antimicrobial agent a No. of isolates tested b N. brasiliensis TET MINO DOXY DMG-DMDOT N. farcinica TET MINO DOXY DMG-DMDOT N. nova TET MINO DOXY DMG-DMDOT N. otitidiscaviarum TET MINO DOXY DMG-DMDOT N. asteroides complex (type VI) TET MINO DOXY DMG-DMDOT b Note that MIC 50 s and MIC 90 s were not computed when the number of isolates tested was less than 10. DMDOT were not determined for all isolates. Both of these antimicrobial agents were highly active against all four groups of RGM. DMG-MINO MICs were 1 g/ml for Tet r isolates of M. fortuitum, M. mucogenicum, M. chelonae, and M. abscessus (Table 2). DMG-DMDOT appeared to be more active than DMG-MINO, with lower MIC 90 s for isolates of the M. fortuitum group (0.125 compared with 1.0 g/ml) and M. abscessus (0.25 compared with 1.0 g/ml). A total of 33 Tet s strains were tested, with 31 being tested against DMG-DM- DOT and 2 being tested against DMG-MINO. The concentration of DMG-DMDOT required to inhibit 50% of the Tet r strains for each RGM species was the same concentration required to inhibit the Tet s isolates of the same species (Table 5). DMG-MINO was tested against too few Tet s isolates to make a similar comparison. MICs for the six ATCC reference strains were comparable to those for the clinical strains (Table 4). The MICs of the three older tetracyclines and DMG-DM- DOT for 68 isolates of Nocardia were compared (Table 3). As expected, the MICs of minocycline and doxycycline were generally four times greater than that of tetracycline, with modal MICs of the former drugs being 2 to 8 g/ml. The MICs were fairly unimodal, with a range of only 3 to 4 dilutions. DMG- DMDOT showed greater activity against two species, N. brasiliensis sensu stricto and N. nova. Although DMG-MINO was tested against only 10 isolates, the results were similar to those obtained with DMG-DMDOT, and some results for DMG- MINO were better than those for DMG-DMDOT (data not shown). The species against which the newer compounds were most active was N. brasiliensis. The MICs of DMG-DMDOT for 20 isolates of N. brasiliensis sensu stricto were all 1 g/ml, and although DMG-MINO was studied against only 2 isolates of N. brasiliensis sensu stricto, the MICs for both isolates were 0.25 g/ml. This activity was 4- to 32-fold greater than that of minocycline or doxycycline. The glycylcyclines had a much wider range of MICs for N. nova than for the other tested. The majority of strains of N. nova and the new taxon N. pseudobrasiliensis were two- to fourfold more susceptible to the glycylcyclines than to minocycline or doxycycline. For three groups of, N. farcinica, N. otitidiscaviarum, and the N. asteroides complex type VI group, the new tetracyclines appeared to have no increased activity compared with those of the older tetracyclines doxycycline and/or minocycline. The MIC range of all of the tetracyclines was 0.5 to 32 g/ml. The MICs for the ATCC reference strains were similar to those for the clinical isolates of the same group (Table 4). TABLE 4. MICs of tetracycline, minocycline, doxycycline, DMG-DMDOT, and DMG-MINO for reference strains of RGM and Strain Tetracycline Minocycline Doxycycline DMG-DMDOT DMG-MINO M. fortuitum group M. fortuitum third biovar ATCC M. fortuitum third biovar ATCC M. peregrinum ATCC M. peregrinum ATCC M. abscessus ATCC M. mucogenicum ATCC N. nova ATCC N. farcinica ATCC N. brasiliensis ATCC N. otitidiscaviarum ATCC N. pseudobrasiliensis ATCC ND a N. pseudobrasiliensis ATCC ND a ND, not determined.

4 VOL. 40, 1996 GLYCYLCYCLINE ACTIVITIES 877 TABLE 5. Comparison of DMG-DMDOT MICs for 140 Tet r and 31 Tet s clinical RGM isolates Tet r isolates Tet s isolates Species No. of isolates No. of isolates Range 50% Range 50% M. fortuitum group a M. chelonae M. abscessus M. mucogenicum a The M. fortuitum group includes M. fortuitum, M. peregrinum, and M. fortuitum unnamed third biovariant complex. DISCUSSION The first members of the tetracycline group were isolated in 1945 from a strain of Streptomyces aureofaciens (25) at Lederle Laboratories. Later, other naturally occurring molecules (like tetracycline and demethylchlorotetracycline) or semisynthetic compounds (like methacycline, doxycycline, and minocycline) were identified (7). In general, these drugs were highly popular because of minimal serious toxicities and because of their broad spectra of activity against many types of gram-positive and gram-negative bacteria including both aerobic and anaerobic bacteria, mycoplasmas, rickettsiae, protozoa, and chlamydiae (7, 25). Additionally, the tetracyclines are active against some groups of RGM, M. marinum, and (8, 9, 11, 35). The more recent spread of plasmid-mediated genes encoding several mechanisms of tetracycline resistance has seriously impaired the routine usefulness of currently available tetracycline compounds (2, 5, 15). Recent modifications have improved the antimicrobial activity and pharmocokinetic properties of the tetracycline and resulted in the production of more favorable analogs (16, 25). Two new derivatives, DMG-MINO and DMG-DMDOT, are undergoing in vitro testing (14, 16, 25, 36). The production of the glycylcyclines represents a significant advance in the tetracyclines in that they are unaffected by the two major genetic mechanisms responsible for the spread of Tet r, i.e., tetracycline efflux, which is the active pumping out of the compound, thereby decreasing the intracellular concentration, and ribosomal protection, whereby tetracyclines no longer bind productively to the bacterial ribosome (12, 25). The latter resistance factor is mediated by a protein classified as TetM; its exact mechanism of protection is still unknown. It may in some manner relate to its ability to hydrolyze GTP in the presence of ribosomes and permit protein synthesis in the presence of tetracycline (16). These new compounds are not only able to inhibit protein synthesis occurring on wild-type ribosomes but can also inhibit protein synthesis occurring on TetM-protected Tet r ribosomes (24). The mechanism of Tet r in RGM,, and streptomyces was totally unknown until recently (20). A study by Pang and colleagues (15) demonstrated that some Tet r strains of these species contain recognized Tet r genes including TetK, TetL, and streptomyces resistance genes known as OtrA, OtrB, and OtrC. Some strains, however, had no recognizable sequences, as determined by hybridization assays with all recognized Tet r genes. The results of the current study suggest that all Tet r in RGM involves resistance mechanisms such as the efflux mechanism that are not effective against the glycylcyclines. Previous studies with glycylcyclines revealed excellent activity against a large number of different bacterial species (14, 24, 25, 36, 37). The only species of mycobacteria previously studied against the glycylcyclines were a few strains of Mycobacterium tuberculosis and Mycobacterium avium complex, both of which were resistant (24, 37). No studies of RGM or Nocardia species have been reported. In our study, the new compounds showed remarkable activity against all species of RGM tested and against some species of Nocardia, thus emphasizing the fact that these compounds have a potential role in the treatment of diseases caused by these organisms. This would appear to be most useful for M. abscessus and M. chelonae, two highly resistant species (including resistance to the older tetracyclines) for which the only potential oral therapy currently is with the newer macrolides (6, 23, 27, 33). ACKNOWLEDGMENTS We thank Joanne Woodring for typing the manuscript, Wyeth- Ayerst Research (formerly Lederle Laboratories) for supplying the glycylcycline powders, and Raymond Testa for support and advice. No financial support was received for this study. REFERENCES 1. Band, J. D., J. I. Ward, D. W. Fraser, N. J. Peterson, V. A. Silcox, R. C. Good, P. R. Ostroy, and J. Kennedy Peritonitis due to Mycobacterium chelonei-like organism associated with intermittent chronic peritoneal dialysis. J. 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