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Antimicrobial Agents and Chemotherapy, October 2002, p. 3164-3167, Vol. 46, No. 10
0066-4804/02/$04.00+0 DOI: 10.1128/AAC.46.10.3164-3167.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.
Comparison of the In Vitro Activity of the Glycylcycline Tigecycline (Formerly GAR-936) with Those of Tetracycline, Minocycline, and Doxycycline against Isolates of Nontuberculous Mycobacteria
Richard J. Wallace Jr.,* Barbara A. Brown-Elliott, Christopher J. Crist, Linda Mann, and Rebecca W. Wilson
Department of Microbiology, University of Texas Health Center, Tyler, Texas
Received 14 March 2002/
Returned for modification 11 April 2002/
Accepted 24 June 2002

ABSTRACT
We compared the in vitro activity of the glycylcycline tigecycline
(formerly GAR-936) with those of tetracycline, doxycycline,
and minocycline by broth microdilution against 76 isolates belonging
to seven species of rapidly growing mycobacteria (RGM) and 45
isolates belonging to five species of slowly growing nontuberculous
mycobacteria (NTM). By using a resistance breakpoint of >4
µg/ml for tigecycline and >8 µg/ml for tetracycline,
all RGM were highly susceptible to tigecycline, with inhibition
of 50% of isolates at

0.12 µg/ml and inhibition of 90%
of isolates at 0.25 µg/ml for
Mycobacterium abscessus and inhibition of both 50 and 90% of isolates at

0.12 µg/ml
for
M. chelonae and the
M. fortuitum group. The MICs of tigecycline
were the same for tetracycline-resistant and -susceptible strains,
and RGM isolates were 4- to 11-fold more susceptible to tigecycline
than to the tetracyclines. In contrast, no slowly growing NTM
were susceptible to tigecycline, and isolates of
M. marinum and
M. kansasii were less susceptible to this agent than to
minocycline. This new antimicrobial offers exciting therapeutic
potential for the RGM, especially for isolates of the
M. chelonae-
M. abscessus group, against which the activities of the currently
available drugs are limited.

INTRODUCTION
Treatment of infections due to rapidly growing mycobacteria
(RGM) remains difficult, in part because of resistance to the
first-line antituberculous agents and also in part because of
resistance to almost all antibacterial agents (
3,
21). The only
drugs with activity against all three major pathogenic species
(
Mycobacterium chelonae,
M. abscessus, and
M. fortuitum) are
amikacin (
13) and clarithromycin (
4). In vitro studies with
prior glycylcyclines (
N,
N-dimethylglycylamido-minocycline [DMG-MINO]
and
N,
N-dimethylglycylamido-6-demethyl-6-deoxytetracycline [DMG-DMDOT])
have shown this class of agents to be extremely active against
this group of organisms, including tetracycline-resistant strains
(
1,
3). The latest glycylcycline, tigecycline (GAR-936), has
shown excellent activity against many tetracycline-resistant
bacterial species (
1,
8,
17; R. N. Jones, A. C. Gales, L. M.
Deshpande, D. M. Johnson, and D. J. Biedenbach, poster 407,
39th Intersci. Conf. Antimicrob. Agents Chemother., 1999). With
this in mind, we undertook a comparative study of the in vitro
susceptibilities of multiple species of nontuberculous mycobacteria
(NTM) to tetracycline, minocycline, doxycycline, and tigecycline.
(A portion of this study was presented at the 41st Interscience Conference on Antimicrobial Agents and Chemotherapy, 16 to 19 December 2001, Chicago, Ill.)

MATERIALS AND METHODS
Organisms.
Clinical mycobacterial isolates submitted to the Mycobacterial/Nocardia
Laboratory at The University of Texas Health Center for susceptibility
testing and selected reference strains were chosen for testing.
Clinical isolates were tested upon receipt in the laboratory,
while the reference strains were taken from frozen stocks stored
at -70°C in Trypticase soy broth with 15% glycerol. This
included 76 clinical isolates of RGM of the
M. fortuitum group
(
n = 26),
M. abscessus (
n = 20),
M. chelonae (
n = 26),
M. immunogenum (
n = 1), and the
M. smegmatis group (
n = 1 isolate each of
M. smegmatis sensu stricto
, M. wolinskyi, and
M. goodii). American
Type Culture Collection (ATCC) reference or type strains of
RGM included
M. fortuitum ATCC 6841
T,
M. peregrinum ATCC 14467
T,
the current NCCLS-recommended susceptibility control strain,
M. peregrinum ATCC 700686 (
10),
M. fortuitum third biovariant
sorbitol-positive strain ATCC 49403 (proposed species
M. neworleansense),
and
M. fortuitum third biovariant sorbitol-negative strain ATCC
49404 (proposed as
M. bonickei) (M. F. Schinsky, M. P. Douglas,
A. G. Steigerwalt, R. W. Wilson, M. M. Floyd, M. I. Daneshvar,
B. A. Brown-Elliott, R. J. Wallace, Jr., M. M. McNeil, D. J.
Brenner, and J. M. Brown, unpublished data).
We also tested 45 isolates of slowly growing NTM that included 11 M. avium complex, 10 M. lentiflavum, 11 M. kansasii, 11 M. marinum, 1 M. xenopi, and 1 M. simiae complex isolates for their susceptibilities to these agents.
Isolates of the M. fortuitum group, M. chelonae, and M. abscessus were identified to the species level by using drug susceptibility patterns (3, 11) or by PCR-restriction enzyme analysis (PRA) of a 439-bp sequence (Telenti fragment) of the 65-kDa hsp gene (12, 15). The remaining RGM species were identified only by PRA (2, 23). Isolates of slowly growing NTM were identified by use of a commercial DNA-RNA probe (Accu-Probe) (Gen-Probe, Inc) or PRA of the 65-kDa hsp gene (15).
Susceptibility testing.
The isolates were tested for their susceptibilities to tetracycline, minocycline, doxycycline, and tigecycline (formerly GAR-936; Wyeth-Ayerst, Pearl River, N.Y.). MICs were determined by broth microdilution and a recently published tentative NCCLS standard method for mycobacterial susceptibility testing of RGM and slowly growing NTM (10). The resistance breakpoints were
16 µg/ml for tetracycline, doxycycline, and minocycline (9). For slowly growing NTM, the NCCLS document does not deal with breakpoints for tetracycline, and breakpoints for doxycycline and minocycline are given only for M. marinum. The breakpoints for the three tetracyclines used here are those for bacterial species that grow aerobically (9). The resistance breakpoint tentatively recommended by the manufacturer for tigecycline is
8 µg/ml.
Quality control.
Quality control for the tetracyclines and tigecycline were performed by using Staphylococcus aureus ATCC 29213 and suggested quality control ranges recommended by the NCCLS for the tetracyclines or the manufacturer for tigecycline (9). Acceptable MIC ranges for S. aureus ATCC 29213 are 0.03 to 0.12 µg/ml for tigecycline, 0.12 to 0.5 µg/ml for doxycycline, 0.06 to 0.5 µg/ml for minocycline, and 0.25 to 1 µg/ml for tetracycline. All quality control results were within the acceptable ranges.

RESULTS
Results for the clinical isolates of the common pathogenic species
of the RGM are shown in Tables
1 and
2. In general, the MICs
of tigecycline for all isolates were very low, with comparable
values for tetracycline-susceptible and tetracycline-resistant
strains.
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TABLE 1. MIC ranges, concentrations that inhibits 50 and 90% of strains, and percentage of strains susceptible for tigecycline (GAR-936) and three tetracyclines for 72 isolates of RGMa
|
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TABLE 2. MIC ranges and concentrations of tetracycline, minocycline, and doxycycline and the new glycylcycline tigecycline (GAR-936) that inhibit 50 and 90% of 50 clinical tetracycline-resistanta isolates of RGM
|
For tetracycline-susceptible strains, tigecycline was approximately
4 dilutions more active than the older tetracyclines. For 15
of 16 tetracycline-susceptible
M. fortuitum strains, the tetracycline
MICs were 0.5 to 8.0 µg/ml, with the mode tetracycline
MIC being 0.5 µg/ml. In contrast, for 15 of the 16 isolates
the tigecycline MICs were

0.12 µg/ml, with the mode tigecycline
MIC being

0.06 µg/ml (16-fold more active than tetracycline)
(data not shown). Similar results were seen with
M. chelonae and a single strain of
M. immunogenum. For the four tetracycline-susceptible
isolates tetracycline MICs were 1 to 4 µg/ml (mode, 4
µg/ml), while the tigecycline MICs for the same strains
ranged from

0.06 to 0.25 µg/ml (mode,

0.06 µg/ml)
(data not shown). Minocycline and doxycycline were more active
against the tetracycline-susceptible strains, with modes of

0.12 and

0.25 µg/ml, respectively. For approximately 50%
of the isolates, however, minocycline MICs were in the range
of 0.25 to 4. 0 µg/ml, which were 3 to 6 dilutions higher
than those of tigecycline.
Results for the five RGM reference strains are given in Table 3. Included are MICs of two glycylcyclines for these strains published previously (3). The activities of tigecycline against these strains were the same as those of DMG-DMDOT and comparable to those of DMG-MINO, with results similar to those achieved for the clinical isolates. This is in contrast to the results for staphylococcal species, against which minocycline is 1 to 4 dilutions more active than tigecycline (1).
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TABLE 3. MICs of tetracycline, minocycline, doxycycline DMG-DMDOT, DMG-MINO, and tigecycline for reference strains of RGM
|
All 33 strains of six species of slowly growing NTM were resistant
to tigecycline (MICs, 16 to >32 µg/ml) (Table
4). Minocycline
was more active than tigecycline against the closely related
photochromogenic species
M. marinum and
M. kansasii. Single
isolates each of
M. xenopi and
M. simiae were also resistant
to tigecycline (MICs, 16 and >32 µg/ml, respectively)
(data not shown in Table
4). Additional numbers of test isolates
of the latter two species are needed to establish the apparent
lack of activity of tigecycline.
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TABLE 4. MIC ranges, concentrations that inhibits 50 and 90% of strains tested, and percentage of strains susceptible for tigecycline (GAR-936) and three tetracyclines against 43 isolates of three species of slowing growing mycobacteriaa
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DISCUSSION
The tetracyclines have been highly popular for antimicrobial
therapy because of their activities against numerous bacterial
organisms, although their use has declined dramatically in the
past decade because of drug resistance. Some of the clinical
isolates against which tetracyclines exhibit activities include
some species of RGM such as
M. fortuitum,
M. smegmatis, and
M. mucogenicum (
6,
20,
21,
22), 50% or more clinical isolates
of which are susceptible. However, at clinically achievable
levels these agents are less effective against other common
pathogenic species of RGM such as
M. chelonae (26% of isolates
are susceptible to doxycycline at 4 µg/ml) and
M. abscessus (4% of isolates are susceptible to doxycycline at 4 µg/ml)
(
13,
18). Preliminary studies suggest that these species contain
a number of resistance determinants that likely play a role
in this resistance (
11).
The introduction of the glycylcyclines represents a significant advance in the tetracyclines, as the glycylcyclines are unaffected by the major genetic mechanisms (efflux, ribosomal protection) responsible for tetracycline resistance among bacterial species (14, 16). Previous studies with two early glycylcyclines, DMG-MINO and DMG-DMDOT, showed that they have excellent in vitro activities against all species of RGM including the members of the M. fortuitum group, M. chelonae, M. abscessus, and M. mucogenicum (3). These results were comparable to the results obtained in the present study with tigecycline (3). The MIC at which 50% of isolates are inhibited (MIC50) and the MIC90 of DMG-DMDOT for the 30 isolates of the M. fortuitum group were
0.06 and 0.125 µg/ml, respectively, values which are the same as those for the 26 isolates tested in the present study against tigecycline. The MICs of the three glycylcyclines for the M. fortuitum reference strains (Table 3) are also similar or identical. For 60 isolates of M. abscessus, the MIC50 and MIC90 of DMG-DOT were 0.125 and 0.25 µg/ml, respectively (3), values which are also the same as those of tigecycline for the 20 isolates tested in the present study.
The activity of tigecycline could be highly useful clinically, especially against RGM species causing serious disease, before susceptibilities are available. Amikacin is the only agent with activity against all RGM species, but it has major renal and auditory toxicities. Tigecycline also has much anticipated usefulness against M. abscessus isolates causing chronic lung disease. The latter disease is incurable with currently available drugs (19). It is increasingly problematic in patients with cystic fibrosis, in whom subsequent transplantation with its associated immune suppression can result in disseminated disease (5, 7).
In contrast, tigecycline appears to have no in vitro activity against any of the species of slowly growing NTM studied, including those species (M. marinum, M. kansasii) that were minocycline susceptible at the currently suggested resistance breakpoints. Experience with MIC testing and specific MIC breakpoints is limited for many slowly growing species, however, especially less commonly encountered species such as M. xenopi and M. simiae. This shortcoming will require additional future studies, an observation recognized in the recent NCCLS document on mycobacterial susceptibility testing (10).

ACKNOWLEDGMENTS
This study was supported in part by a grant from Wyeth-Ayerst.
We gratefully acknowledge Wyeth-Ayerst Research and Raymond
Testa for financial support and advice.
We also thank Joanne Woodring for preparation of the manuscript.

FOOTNOTES
* Corresponding author. Mailing address: Department of Microbiology at the University of Texas Health Center, 11937 U.S. Hwy. 271, Tyler, TX 75708. Phone: (903) 877-7680. Fax: (903) 877-7652. E-mail:
richard.wallace{at}uthct.edu.


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Antimicrobial Agents and Chemotherapy, October 2002, p. 3164-3167, Vol. 46, No. 10
0066-4804/02/$04.00+0 DOI: 10.1128/AAC.46.10.3164-3167.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.
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